Protein a compositions and methods of use

ABSTRACT

The invention provides methods for modulating an immune response in a subject including, for example, administering to the subject a composition comprising an effective amount of a lymphocyte differentiation factor sufficient to modulate the immune response. In one aspect, the lymphocyte differentiation factor comprises protein A (PA).

TECHNICAL FIELD

[0001] The invention relates to immune response modulation and treatingimmune disorders and pathologies associated with or caused by immunedisorders.

BACKGROUND

[0002] Protein A is a 40,000 Da glycoprotein extracted from the cellwall of various bacteria. Bacteria use PA as a targeting and bindingsite for tissue attachment. Protein A has a high affinity for the Fcportion of certain immunoglobulin classes and even higher affinity forthose immunoglobulins once they have bound antigen. This biochemicalproperty of PA has been used in a large number of applications. Theseapplications of PA reflect a use of the Fc binding properties of themolecule or PA's ability to stimulate humoral immunity in the absence ofspecific antigen induction (Superantigen applications).

SUMMARY

[0003] The invention is based at least in part on a feature(s) of PAthat is distinct from its Fc binding characteristics and Superantigenproperties. This feature confers one or more of the following activitiesin animals: an ability to re-regulate aberrant process(es) and inhibittissue damage or reverse at least a portion of existing tissue damagecaused by the unregulated process(es); an ability to re-regulateaberrant or undesirable immune process(es).

[0004] The invention therefore provides methods for modulating an immuneresponse in a subject. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of a lymphocyte differentiation factor sufficient to modulate theimmune response. In one aspect, the lymphocyte differentiation factorcomprises protein A (PA).

[0005] Also provided are methods for treating an immune dysfunction in asubject with or at risk of an immune dysfunction. In one embodiment, amethod includes administering to the subject a composition comprising aneffective amount of protein A (PA) sufficient to treat the immunedysfunction. In one aspect, the immune dysfunction comprises anautoimmune disorder (e.g., rheumatoid arthritis, juvenile rheumatoidarthritis, osteoarthritis, psoriatic arthritis, diabetes mellitus,multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupuserythematosis (SLE), autoimmune thyroiditis, atopic dermatitis,eczematous dermatitis, psoriasis, Sjögren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitisposterior, interstitial lung fibrosis, Hashimoto's thyroiditis,autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus,insulin-resistant diabetes mellitus, immune-mediated infertility,autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus,dermatitis herpetiformis, autoimmune alopecia, Vitiligo, autoimmunehemolytic anemia, autoimmune thrombocytopenic purpura, perniciousanemia, Guillain-Barre syndrome, Stiff-man syndrome, acute rheumaticfever, sympathetic ophthalmia, Goodpasture's syndrome, systemicnecrotizing vasculitis, antiphospholipid syndrome or an allergy). Inanother aspect, the immune dysfunction comprises an immunodeficiency(e.g., severe combined immunodeficiency (SCID) such as recombinaseactivating gene (RAG 1/2) deficiency, adenosine deaminase (ADA)deficiency, interleukin receptorsy chain (γc) deficiency,Janus-associated kinase 3 (JAK3) deficiency and reticular dysgenesis;primary T cell immunodeficiency such as DiGeorge syndrome, Nudesyndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiencyand purine nucleotide phosphorylase (PNP) deficiency; predominantlyantibody deficiencies such as X-linked agammaglobulinemia (Bruton'styrosine kinase deficiency); autosomal recessive agammaglobulinemia suchas Mu heavy chain deficiency; surrogate light chain (γ5/14.1)deficiency; Hyper-IgM syndrome either X-linked (CD40 ligand deficiency)and others; Ig heavy chain gene deletion; IgA deficiency; deficiency ofIgG subclasses (with or without IgA deficiency); common variableimmunodeficiency (CVID); antibody deficiency with normalimmunoglobulins; transient hypogammaglobulinemia of infancy; interferonγ receptor (IFNGR1, IFNGR2) deficiency; interleukin 12 and interleukin12 receptor deficiency; immunodeficiency with thymoma; Wiskott-Aldrichsyndrome (WAS protein deficiency); ataxia telangiectasia (ATMdeficiency); X-linked lymphoproliferative syndrome (SH2D1A/SAPdeficiency); and hyper IgE syndrome). In yet another aspect, the immunedysfunction comprises an immunodeficiency associated with or secondaryto another disease (e.g., chromosomal instability or defective repairsuch as Bloom syndrome, Xeroderma pigmentosum, Fanconi anemia, ICFsyndrome, Nijmegen breakage syndrome and Seckel syndrome; chromosomaldefects such as Down syndrome (Trisomy 21), Turner syndrome andDeletions or rings of chromosome 18 (18p- and 18q-); skeletalabnormalities such as short-limbed skeletal dysplasia (short-limbeddwarfism) and cartilage-hair hypoplasia (metaphyseal chondroplasia);Immunodeficiency associated with generalized growth retardation such asSchimke immuno-osseous dysplasia, Dubowitz syndrome, Kyphomelicdysplasia with SCID, Mulibrey's nannism, Growth retardation, facialanomalies and immunodeficiency and Progeria (Hutchinson-Gilfordsyndrome); immunodeficiency with dermatologic defects such asectrodactyly-ectodermal dysplasia-clefting syndrome, immunodeficiencywith absent thumbs, anosmia and ichthyosis, partial albinism,Dyskeratosis congenita, Netherton syndrome, Anhidrotic ectodermaldysplasia, Papillon-Lefevre syndrome and congenital ichthyosis;hereditary metabolic defects such as acrodermatitis enteropathica,transcobalamin 2 deficiency, type 1 hereditary orotic aciduria,intractable diarrhea, abnormal facies, trichorrhexis andimmunodeficiency, methylmalonic acidemia, biotin dependent carboxylasedeficiency, mannosidosis, glycogen storage disease, type 1b,Chediak-Higashi syndrome; hypercatabolism of immunoglobulin such asfamilial hypercatabolism, intestinal lymphangiectasia; chronicmuco-cutaneous candidiasis; hereditary or congenital hyposplenia orasplenia; and Ivermark syndrome).

[0006] Further provided are methods for reducing an inflammatoryresponse in a subject with or at risk of an inflammatory response. Inone embodiment, a method includes administering to the subject acomposition comprising an effective amount of protein A (PA) sufficientto reduce an inflammatory response. In one aspect, the inflammatoryresponse is chronic or acute. In another aspect, the inflammatoryresponse is at least in part mediated by an antibody (e.g., one or moreauto-antibodies) or at least in part mediated by cellular immunity.

[0007] Additionally provided are methods for reducing inflammation in asubject. In one embodiment, a method includes administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to reduce the inflammation. In one aspect, the inflammationis chronic or acute. In another aspect, the inflammation is at least inpart antibody or cell mediated. In still another aspect, the treatmentresults in a reduction in severity of a symptom of inflammation (e.g.,swelling, pain, headache, fever, nausea, skeletal joint stiffness, ortissue or cell damage). In yet another aspect, the treatment results ininhibition of antibody production or lymphoid cell proliferation.

[0008] Further provided are methods for inhibiting tissue or cell damagein a subject caused by an inflammatory response or inflammation. In oneembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to treatinhibiting tissue or cell damage caused by an inflammatory response orinflammation. In one aspect, the tissue or cell damage is caused by achronic or acute inflammatory response or inflammation. In anotheraspect, the inflammatory response or inflammation is at least in partantibody or cell mediated. In yet another aspect, the tissue or celldamage is present in thymus, liver, kidney, spleen, skin, or a skeletaljoint (e.g., knee, ankle, hip, shoulder, wrist, finger, toe, or elbow).In still another aspect, the treatment results in inhibiting orpreventing further tissue or cell damage.

[0009] Methods for treating existing tissue or cell damage in a subjectcaused by an inflammatory response or inflammation are provided. In oneembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to treatexisting tissue or cell damage caused by an inflammatory response orinflammation. In one aspect, the existing tissue or cell damage iscaused by a chronic or acute inflammatory response or inflammation. Inanother aspect, the inflammatory response or inflammation is at least inpart antibody or cell mediated. In yet another aspect, the existingtissue or cell damage is present in thymus, liver, kidney, spleen, skin,or a skeletal joint (e.g., knee, ankle, hip, shoulder, wrist, finger,toe, or elbow). In still other aspects, the treatment results inreversing tissue or cell damage or results in inhibiting or preventingfurther tissue or cell damage.

[0010] Methods of treating splenomegalia in a subject are also provided.In one embodiment, a method includes administering to the subject acomposition comprising an effective amount of protein A (PA) sufficientto treat splenomegalia.

[0011] Methods of inhibiting proliferation or survival of a splenocytein a subject having or at risk of having undesirable splenocyteproliferation or survival are additionally provided. In one embodiment,a method includes administering to the subject a composition comprisingan effective amount of protein A (PA) sufficient to inhibitproliferation or survival of the splenocyte.

[0012] Methods of stimulating differentiation or apoptosis of asplenocyte in a subject having or at risk of having undesirablesplenocyte proliferation or apoptosis are further provided. In oneembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to stimulatedifferentiation or apoptosis of the splenocyte.

[0013] Methods of reducing antibody production by a splenocyte in asubject having or at risk of having undesirable numbers of an antibodyare provided. In one embodiment, a method includes administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to reduce antibody (e.g., auto-antibody) production by asplenocyte.

[0014] Methods of reducing numbers of an antibody producing splenocytein a subject having or at risk of having undesirable numbers of thesplenocytes are also provided. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to reduce the antibody (e.g.,auto-antibody) producing splenocyte.

[0015] Methods of reducing natural killer (NK) cell cytotoxicity in asubject having or at risk of having undesirable NK cell cytotoxicity areadditionally provided. In one embodiment, a method includesadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to reduce undesirable NK cellcytotoxicity.

[0016] Methods of inhibiting rejection of a transplanted cell, tissue ororgan in a subject are further provided. In one embodiment, a methodincludes administering to the subject a composition comprising aneffective amount of protein A (PA) sufficient to inhibit rejection of atransplanted cell, tissue or organ (e.g., an allograft or xenograft). Invarious aspects, PA is administered prior to, substantiallycontemporaneously with, or following transplanting the cell, tissue ororgan.

[0017] Methods of stimulating differentiation of lymphoid cells areprovided. In one embodiment, a method includes contacting one or morelymphoid cells in vitro, ex vivo or in vivo with a compositioncomprising an effective amount of protein A (PA) sufficient to stimulatedifferentiation of one or more lymphoid cells. In various aspects, thelymphoid cell is a T or B cell.

[0018] The invention is also based at least in part on the low amountsof PA required to achieve the activities. In particular, PA has theaforementioned activities at low concentrations, typically less thanamounts used for Superantigen applications.

[0019] The invention methods therefore may be practiced with PA inamounts effective to elicit one or more of the activities disclosedherein, but without substantial superantigen activity, Fc bindingactivity or substantially stimulating humoral immunity. In oneembodiment, an amount is a dose of about 1 picogram to about 1 microgramof PA. In another embodiment, an amount is a single dose of about 1picogram to about 1 microgram of PA administered intermittently overabout 1 to 15 weeks. In yet another embodiment, an amount is a singledose of about 1 picogram to about 1 microgram administered of PA onalternating days over about 7 to 21 days.

[0020] Furthermore, the invention provides compositions that elicit oneor more of the activities disclosed herein in unit dosage form. In oneembodiment, a composition comprises a unit dosage form of PA from about0.5-5, 5-10, 10-20, 20-50 or 50-100, 100-500, 100-1000 picograms. Inanother embodiment, a composition comprises a unit dosage form of PAfrom about 1-10, 10-100, 100-500 or about 500-1000 nanograms. In yetanother embodiment, a composition comprises a unit dosage form of PAsufficient to reduce an inflammatory response or inflammation in asubject.

[0021] Pharmaceutical compositions are provided that include a unitdosage form of PA (e.g., 0.5-5, 5-10, 10-20, 20-50 or 50-100, 100-500,100-1000 picograms; 1-10, 10-100, 100-500 or about 500-1000 nanograms).Pharmaceutical compositions are provided that include a unit dosage formof PA that elicits one or more of the activities disclosed herein (e.g.,reduces an inflammatory response or inflammation in a subject).

[0022] Kits including a unit dosage form of PA (or pharmaceuticalcompositions) are also provided, such kits optionally further includinginstructions for use in a method of the invention (e.g., reducing aninflammatory response, inflammation or tissue or cell damage caused byan inflammatory response or inflammation in a subject). In oneembodiment, a kit includes a plurality of unit dosage forms of PA. Inanother embodiment, a kit further includes a drug (e.g., that reduces aninflammatory response or inflammation).

DESCRIPTION OF DRAWINGS

[0023]FIG. 1 shows the weight gain and growth kinetics of A) controluntreated normal mice (C57BL/6J); B) untreated BXSB mice; and C) BXSBmice with PA treatment.

DETAILED DESCRIPTION

[0024] The invention is based at least in part on the characterizationof one or more activities of protein A (PA) that appear to be distinctfrom its Superantigen properties, Fc binding activity or its ability tostimulate humoral immunity. These distinct PA activities are believed tobe at least in part attributable to PAs ability to re-regulate ornormalize undesirable or aberrant physiological process(es) such asimmune dysfunction. PA's ability to re-regulate or normalizephysiological process(es) results in many different beneficialactivities including, for example, modulating aberrant or undesirableimmune response (e.g., re-regulating or normalizing), ameliorating orreducing autoimmunity, reducing inflammation or an inflammatoryresponse, inhibiting or reversing at least a portion of tissue damagecaused by an un-regulated process(es) such as an undesirable or aberrantimmune response.

[0025] More particularly, PA efficacy is demonstrated through the use ofa collagen induced arthritis (CIA) murine inflammation model. Theinduced immune response to Type II collagen is antibody mediated causinga rapidly progressing inflammatory response which can be assessed bymeasuring the inflammation in affected joints and also by applying astandardized clinical assessment for the affected joints (termed“clinical index” or “CI”). The CI assessment involves both swelling andmobility measures. As shown in Example 1, PA at low concentrationsinhibits an acute inflammatory response in the CIA murine model.Histological examination of knee and ankle joints revealed a reductionin tissue damage as well as of immune cell infiltration of the synovium.

[0026] PA efficacy is also demonstrated in BXSB animal model, whichrepresents a combined autoimmune deficiency disease having a geneticbasis that results in early death of the male animals. As shown inExamples 3 to 8, PA at low concentrations modifies many diseasecharacteristics in the BXSB animal, in many cases re-regulating thevarious manifestations of the disease (cellular and histological)towards base-line levels (i.e., towards normalization). For example, PAinhibits or prevents the early onset of the wasting (weight loss);regulates expansion of the splenic compartment; inhibits over-expressionor -activity of humoral immunity; inhibits over-expression or -activityof cellular immunity; modulates differentiation of cells of lymphoidcell lineage; and ameliorates, reduces or reverses tissue damage causedby or associated with the disease processes. The data further indicatesthat PA has the same dose response pattern as in the CIA model.

[0027] Thus, PA activities at low concentrations include, for example,one or more of regulating expansion of the splenic compartment(modulating proliferation, apoptosis or differentiation), regulatingaberrant or undesirable humoral immunity (inhibiting autoantibodyproduction or inhibiting cells that produce autoantibodies), regulatingaberrant or undesirable cellular immunity (normalizing TH₁/TH₂ balance,inhibiting cytotoxicity responses), modulating proliferation, apoptosis,or differentiation of cells within the lymphoid cell lineage (e.g.,normalizing T cell populations such as increasing numbers of mature Tcells, e.g., CD69-CD4+), inhibiting or reversing cell or tissue damagecaused by undesirable or aberrant immune response (inhibiting orpreventing disease progression, promoting or enhancing disease reversalor tissue regeneration), and normalizing T or B splenocyte numbers ortheir response to one or more mitogens.

[0028] PA is therefore useful in treating a subject in need of one ormore of the aforementioned activities associated with PA. The inventiontherefore provides, inter alia, methods for modulating an immuneresponse (cellular or humoral), methods for treating an undesirable oraberrant immune response (e.g., immune dysfunction) and methods forinhibiting, preventing or reversing a physiological effect caused by orassociated with an immune response in a subject. In one embodiment, amethod includes administering to a subject a composition comprising aneffective amount of a lymphocyte differentiation factor sufficient tomodulate the immune response. In another embodiment, a method includesadministering to a subject a composition comprising an effective amountof PA sufficient to modulate the immune response.

[0029] As used herein, the term “modulate” means a detectable change inan activity or function or effect to which the term is referring.Modulate can mean any increase, decrease, reduction, inhibition,prevention, stimulation, promotion, enhancement in the activity orfunction or effect to which the term refers. For example, modulating animmune response means that activity or function or an effect of theimmune response is detectably changed, e.g., an increase, decrease,reduction, inhibition, prevention, stimulation, promotion, orenhancement of humoral or cell mediated immunity. Changes in an immuneresponse indicative of modulation, including, for example, numbers of Tand B cells, proliferation, apoptosis, differentiation, cytotoxicity,antibody production or numbers of antibody producing cells (e.g.,autoantibodies), mitogen responsiveness, inflammation, cell or tissuedamage, or symptoms thereof, can be measured by a variety of methodsdisclosed herein or known in the art. An “effective amount” or“sufficient amount” means an amount needed to achieve the activity oreffect.

[0030] As used herein, the terms “re-regulate,” “normalize” andgrammatical variations thereof mean a shift towards base line levels. Ashift towards base line levels can include, for example, changes innumbers of cells, differentiation status, antibody production or amountsof antibody (e.g., autoantibodies in circulation), cytotoxicity orresponse to a mitogen. Thus, to re-regulate or normalize numbers ofsplenocytes in BXSB spleen, for example, means a return towards thenumber of splenocytes typically found in a normal (e.g., disease free)animal spleen, e.g., C57BL/6. Likewise, to re-regulate or normalizeautoantibodies means to reduce the amount of such antibodies to thosemore typically found in a normal (e.g., disease free) animal. Tore-regulate or normalize populations of T cells in BXSB means, forexample, to shift the T cell population towards that typically observedin C57BL/6, e.g., a change from immature to a mature T cell population.

[0031] The amount of re-regulation or normalization that can occur canbe a return to at or near baseline levels typical for a normal animal(within 5-25% of baseline), but may be less, for example, a detectableshift towards baseline levels even though the shift does not return thelevels to at or near baseline (e.g., within 25-100% or 25-200% ofbaseline). The shift will depend on the extent of deviation frombaseline in the untreated state, the amount of PA administered and whatis being returned to baseline. For example, splenocyte numbers for BXSBare 5 to 6-times greater than C57BL/6. A re-regulation or normalizationof splenocyte numbers for BXSB would therefore mean that splenocytenumbers were reduced in BXSB following treatment. For example, areduction from 5 to 6-times greater than C57BL/6 mice to 1 to 3-timesgreater than C57BL/6 mice, or more, such as within about 10-50% ofsplenocyte numbers typically observed in C57BL/6 mice. Similarly, inBXSB there is a 200% increase of ANA at 5 weeks and a 1000% increase ofANA at 11 weeks. A re-regulation or normalization of auto-antibodies forBXSB would therefore mean that auto-antibody numbers (e.g., ANA) werereduced following treatment. For example, treatment with 0.01 μg PAreturned these values to at or near baseline (e.g., within 25% ofbaseline). Thus, autoantibody numbers may decrease from 10-times greaterthan C57BL/6 mice to 5 to 8-times greater than C57BL/6 mice or to 1 to5-times greater than C57BL/6 mice, or more, such as within about 10-50%of autoantibody numbers in C57BL/6 mice.

[0032] The invention further provides, inter alia, methods for treatingan immune dysfunction in a subject with or at risk of an immunedysfunction. In one embodiment, a method includes administering to asubject a composition comprising an effective amount of protein A (PA)sufficient to treat the immune dysfunction. In one aspect, the immunedysfunction comprises an autoimmune disorder.

[0033] As used herein, “immune dysfunction” or “immune disorder” meansan undesirable immune response, function or activity, that is greaterthan (e.g., autoimmunity) or less than (e.g., immunodeficiency) desired.An undesirable immune response, function or activity can be a normalresponse, function or activity. Thus, normal immune responses that arenot considered aberrant so long as they are undesirable are includedwithin the meaning of these terms. An undesirable immune response,function or activity can also be an abnormal response, function oractivity. An abnormal or an aberrant immune response, function oractivity deviates from normal. Immune dysfunction or disorder can beprimarily humoral or cellular in nature, or both, either chronic oracute.

[0034] Immune dysfunction or disorders include disorders characterizedby many different physiological symptoms or abnormalities. As disclosedherein, BXSB mouse model is an immune disorder characterized by a vastarray of physiological symptoms and abnormalities which can be treatedin accordance with the invention (see for example, Examples 4 to 9). Theinvention is therefore useful in treating any immune dysfunction ordisorder characterized by many different physiological symptoms andabnormalities including disorders having one or more physiologicalsymptoms or abnormalities similar to BXSB mouse model, or equivalentdisorders in different species. For example, BXSB mouse is characterizedby aberrant splenocyte proliferation, apoptosis or differentiation whichleads to expansion of the splenic compartment and a consequent increasein numbers of immature splenocytes. Thus, although the particular typesof splenocytes whose numbers increase in BXSB mouse may be differentthan those of another species with an immune disorder (e.g., withrespect to their CD markers), the invention is applicable to anydisorder characterized as having undesirable numbers of immaturesplenocytes (caused by excess cell proliferation, survival or failure ofapoptosis) or decreased numbers of mature splenocytes in a subject.

[0035] Thus, as the invention is useful for re-regulating or normalizingmany facets of an immune response, which leads to ameliorating orreducing one or more of the many different symptoms and abnormalities ofthe immune disorder, the invention is broadly applicable to disordersthat are different from that which occurs in BXSB mouse. Of course,disorders treatable in accordance with the invention include thosecharacterized as having one or more characteristics, symptoms orabnormalities of BXSB even if less severe than those present in BXSBmouse.

[0036] Particular examples of immune disorders to which the inventionapplies include autoimmune disorders and immunodeficiencies. Autoimmunedisorders are generally characterized as an undesirable or aberrantresponse, activity or function of the immune system. Immunodeficienciesare generally characterized by decreased or insufficient humoral orcell-mediated immune responsiveness or memory, or increased orundesirable tolerance. Such disorders that may be treated in accordancewith the invention include but are not limited to disorders that causecell or tissue/organ damage in the subject.

[0037] Thus, the invention additionally provides, inter alia, methodsfor treating an autoimmune disorder in a subject with or at risk of anautoimmune disorder. In one embodiment, a method includes administeringto a subject a composition comprising an effective amount of protein A(PA) sufficient to treat the autoimmune disorder. In various aspects,the autoimmune disorder comprises rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis, diabetesmellitus, multiple sclerosis, encephalomyelitis, myasthenia gravis,systemic lupus erythematosis (SLE), autoimmune thyroiditis, atopicdermatitis, eczematous dermatitis, psoriasis, Sjögren's Syndrome,Crohn's disease, aphthous ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior,interstitial lung fibrosis, Hashimoto's thyroiditis, autoimmunepolyglandular syndrome, insulin-dependent diabetes mellitus,insulin-resistant diabetes mellitus, immune-mediated infertility,autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus,dermatitis herpetiformis, autoimmune alopecia, Vitiligo, autoimmunehemolytic anemia, autoimmune thrombocytopenic purpura, perniciousanemia, Guillain-Barre syndrome, Stiff-man syndrome, acute rheumaticfever, sympathetic ophthalmia, Goodpasture's syndrome, systemicnecrotizing vasculitis, antiphospholipid syndrome or an allergy.

[0038] The invention additionally provides, inter alia, methods fortreating immunodeficiency in a subject with or at risk of animmunodeficiency. In one embodiment, a method includes administering toa subject a composition comprising an effective amount of protein A (PA)sufficient to treat the immunodeficiency. In various aspects, theimmunodeficiency comprises severe combined immunodeficiency (SCID) suchas recombinase activating gene (RAG 1/2) deficiency, adenosine deaminase(ADA) deficiency, interleukin receptor y chain (γc) deficiency,Janus-associated kinase 3 (JAK3) deficiency and reticular dysgenesis;primary T cell immunodeficiency such as DiGeorge syndrome, Nudesyndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiencyand purine nucleotide phosphorylase (PNP) deficiency; predominantlyantibody deficiencies such as X-linked agammaglobulinemia (Bruton'styrosine kinase deficiency); autosomal recessive agammaglobulinemia suchas Mu heavy chain deficiency; surrogate light chain (γ5/14.1)deficiency; Hyper-IgM syndrome either X-linked (CD40 ligand deficiency)and others; Ig heavy chain gene deletion; IgA deficiency; deficiency ofIgG subclasses (with or without IgA deficiency); common variableimmunodeficiency (CVID); antibody deficiency with normalimmunoglobulins; transient hypogammaglobulinemia of infancy; interferonγ receptor (IFNGR1, IFNGR2) deficiency; interleukin 12 and interleukin12 receptor deficiency; immunodeficiency with thymoma; Wiskott-Aldrichsyndrome (WAS protein deficiency); ataxia telangiectasia (ATMdeficiency); X-linked lymphoproliferative syndrome (SH2D1A/SAPdeficiency); and hyper IgE syndrome). In yet another aspect, the immunedysfunction comprises an immunodeficiency associated with or secondaryto another disease (e.g., chromosomal instability or defective repairsuch as Bloom syndrome, Xeroderma pigmentosum, Fanconi anemia, ICFsyndrome, Nijmegen breakage syndrome and Seckel syndrome; chromosomaldefects such as Down syndrome (Trisomy 21), Turner syndrome andDeletions or rings of chromosome 18 (18p- and 18q-); skeletalabnormalities such as short-limbed skeletal dysplasia (short-limbeddwarfism) and cartilage-hair hypoplasia (metaphyseal chondroplasia);Immunodeficiency associated with generalized growth retardation such asSchimke immuno-osseous dysplasia, Dubowitz syndrome, Kyphomelicdysplasia with SCID, Mulibrey's nannism, Growth retardation, facialanomalies and immunodeficiency and Progeria (Hutchinson-Gilfordsyndrome); immunodeficiency with dermatologic defects such asectrodactyly-ectodermal dysplasia-clefting syndrome, immunodeficiencywith absent thumbs, anosmia and ichthyosis, partial albinism,Dyskeratosis congenita, Netherton syndrome, Anhidrotic ectodermaldysplasia, Papillon-Lefevre syndrome and congenital ichthyosis;hereditary metabolic defects such as acrodermatitis enteropathica,transcobalamin 2 deficiency, type 1 hereditary orotic aciduria,intractable diarrhea, abnormal facies, trichorrhexis andimmunodeficiency, methylmalonic acidemia, biotin dependent carboxylasedeficiency, mannosidosis, glycogen storage disease, type 1b,Chediak-Higashi syndrome; hypercatabolism of immunoglobulin such asfamilial hypercatabolism, intestinal lymphangiectasia; chronicmuco-cutaneous candidiasis; hereditary or congenital hyposplenia orasplenia; or Ivermark syndrome.

[0039] Additional particular examples of immune dysfunction or disordersto which the invention applies include an undesirable or aberrantinflammatory response or inflammation. Such disorders may be mediated bycellular or humoral immunity, or a combination of both.

[0040] The invention therefore also provides, inter alia, methods forreducing or inhibiting an inflammatory response or inflammation (chronicor acute) in a subject with or at risk of an inflammatory response orinflammation. In one embodiment, a method includes administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to reduce or inhibit an inflammatory response. In anotherembodiment, a method includes administering to the subject a compositioncomprising an effective amount of protein A (PA) sufficient to reduce orinhibit inflammation. In one aspect, the inflammatory response orinflammation is at least in part mediated by an antibody (e.g., one ormore autoantibodies). In another aspect, the inflammatory response orinflammation is at least in part mediated by cellular immunity. Invarious aspects, a method (e.g., treatment) results in a reduction inseverity or frequency of a symptom of an inflammatory response orinflammation. In particular aspects, the symptom includes one or more ofswelling, pain, headache, fever, nausea, skeletal joint stiffness, ortissue or cell damage. In additional particular aspects, a method (e.g.,treatment) results in inhibition of antibody production or lymphoid cellproliferation.

[0041] Immune dysfunction, for example, undesirable or aberrantinflammation or an inflammatory response may cause, directly orindirectly, cell or tissue/organ damage, either to multiple cells,tissues or organs, or specifically to a single cell type, organ ortissue type. For example, as disclosed in the Examples, CIA and BXSBmodels exhibited damage in multiple tissues, as evidenced by changes inhistology. Tissues that exhibited damage included knee, ankle, thymus,kidney and liver. Treatment in accordance with the invention resulted inat least a partial reversal of existing tissue damage or a regenerationof normal tissue (see, for example, Tables 9 and 10).

[0042] The invention therefore also provides, inter alia, methods fortreating, inhibiting and reversing tissue or cell damage, and promotingor enhancing tissue or cell regeneration in a subject caused by immunedysfunction (e.g., an undesirable or aberrant inflammatory response orinflammation). In one embodiment, a method includes administering to asubject a composition comprising an effective amount of protein A (PA)sufficient to treat existing tissue or cell damage caused by immunedysfunction (e.g., an undesirable or aberrant inflammatory response orinflammation). In another embodiment, a method includes administering toa subject a composition comprising an effective amount of protein A (PA)sufficient to inhibit tissue or cell damage (existing or prophylaxis)caused by immune dysfunction (e.g., a chronic or acute undesirable oraberrant inflammatory response or inflammation). In yet anotherembodiment, a method includes administering to a subject a compositioncomprising an effective amount of protein A (PA) sufficient to reverseexisting tissue or cell damage caused by immune dysfunction (e.g., anundesirable or aberrant inflammatory response or inflammation). In stillanother embodiment, a method includes administering to a subject acomposition comprising an effective amount of protein A (PA) sufficientto promote or enhance tissue or cell regeneration caused by immunedysfunction (e.g., an undesirable or aberrant inflammatory response orinflammation). In one aspect, the inflammatory response or inflammationis at least in part mediated by an antibody (e.g., one or moreautoantibodies). In another aspect, the inflammatory response orinflammation is at least in part mediated by cellular immunity. In yetother aspects, the tissue damage is present in thymus, liver, kidney,spleen, skin, or a skeletal joint. In particular aspects, tissue damagein a skeletal joint is present in knee, ankle, hip, shoulder, wrist,finger, toe, or elbow.

[0043] Methods of the invention include treatment methods that inhibitor prevent further tissue or cell damage. Thus, the invention alsoprovides methods of treating existing tissue or cell damage in a subjectcaused by immune dysfunction (e.g., an undesirable or aberrantinflammatory response or inflammation), as well as inhibiting orpreventing further tissue or cell damage. In one embodiment, a methodincludes administering to the subject a composition comprising aneffective amount of protein A (PA) sufficient to inhibit or preventfurther tissue or cell damage caused by immune dysfunction (e.g., anundesirable or aberrant inflammatory response or inflammation). Examplesof existing damage treatable in accordance with the invention include,for example, tissue or organ damage. Exemplary damage as disclosedherein is present in thymus, liver, kidney, spleen, skin, or a skeletaljoint (e.g., knee or ankle).

[0044] Methods of the invention that include treatment of aninflammatory response or inflammation are desired to reduce a symptom orcharacteristic of an inflammatory response or inflammation. At the wholebody level, an inflammatory response or inflammation is generallycharacterized by swelling, pain, headache, fever, nausea, skeletal jointstiffness or lack of mobility, redness or other discoloration. At thecellular level, an inflammatory response or inflammation ischaracterized by one or more of cell infiltration of the region,production of antibodies (e.g., autoantibodies), production ofcytokines, lymphokines, chemokines, interferons and interleukins, growthand maturation (e.g., differentiation factors), cell proliferation,differentiation, accumulation or migration and cell, tissue or organdamage. Thus, treatment will reduce, inhibit or prevent one or more ofsymptoms (severity or frequency of occurrence) or characteristics of aninflammatory response or inflammation.

[0045] Methods of the invention also include treating splenomegalia(i.e., enlarged spleen) in a subject. Such methods include administeringto the subject a composition comprising an effective amount of protein A(PA) sufficient to treat splenomegalia. Without being bound by anytheory, treating splenomegalia typically stimulates, increases orpromotes proliferation or survival of mature lymphocytes (e.g., T or Bsplenocytes), or differentiation from immature to mature cells, orinhibits or decreases proliferation or survival of immature cells to aphysiological status more typical of a normal animal, i.e., an animalthat does not exhibit splenomegalia. Accordingly, methods forstimulating, increasing or promoting proliferation or survival of maturelymphocytes (e.g., T or B splenocytes) or differentiation from immatureto mature lymphocytes (e.g., T or B splenocytes), and inhibiting ordecreasing proliferation or survival of immature lymphocytes (e.g., T orB splenocytes), are provided.

[0046] Methods of the invention further include inhibiting, reducing orpreventing antibody production in a subject. In one embodiment, a methodincludes administering to a subject having an undesirable antibody or anaberrant antibody a composition comprising an effective amount ofprotein A (PA) sufficient to reduce antibody production. Autoantibodiesare but one example of an antibody in which it may be desired toinhibit, reduce or prevent its production. Antibody production can beinhibited, reduced or prevented either directly, by causing the cell(e.g., splenocyte) that produces the antibody to reduce antibodyproduction, or indirectly, by reducing numbers of cells (e.g.,splenocytes) that produce the antibody.

[0047] Methods of the invention additionally include inhibiting,reducing or preventing natural killer (NK) cell cytotoxicity in asubject having or at risk of having undesirable NK cell cytotoxicity. Inone embodiment, a method includes administering to a subject acomposition comprising an effective amount of protein A (PA) sufficientto inhibit, reduce or prevent undesirable NK cell cytotoxicity.

[0048] Methods of the invention moreover include stimulating, promotingor enhancing differentiation of a lymphoid cell. In one embodiment, amethod includes contacting a lymphoid cell in vitro, ex vivo or in vivowith a composition comprising an effective amount of protein A (PA)sufficient to stimulate, promote or enhance differentiation of alymphoid cell.

[0049] The term “contacting” means direct or indirect binding orinteraction between two or more entities (e.g., between PA and a cell ormolecule). Contacting as used herein includes in solution, in solidphase, in vitro, in a cell and in vivo.

[0050] Assays for detecting an activity of PA include; cellular changesin lymphocyte numbers, proliferation, apoptosis or survival anddifferentiation include trypan blue exclusion (viability); changes incellular CD markers or other molecules (differentiation); amounts ofantibody (e.g., circulating autoantibodies can be measured using ELISAor other antibody detection assays); tissue or organ improvementincluding inhibiting further damage or reversing existing tissue damage(histology, tissue or organ function, or enzyme levels indicative ofimproved function); whole body effects (weight gain or a decrease inweight loss or wasting, improved mobility); and expansion of spleen(histology, numbers of lymphocytes and their differentiation state) asdisclosed herein and further known in the art.

[0051] As the invention can be used to inhibit, reduce or prevent anundesirable immune response in a subject, further provided are methodsfor inhibiting, reducing or preventing rejection of a transplanted cell,tissue or organ in a subject (i.e., Host v. Graft disease). In oneembodiment, a method includes administering to a subject a compositioncomprising an effective amount of protein A (PA) sufficient to inhibit,reduce or prevent rejection of a transplanted cell, tissue or organ.Exemplary cells include neural cells. Exemplary tissues include skin,blood vessel, eye and bone marrow. Exemplary organs include heart, lung,liver and kidney. In various aspects, PA is administered prior to,substantially contemporaneously with, or following transplanting thecell, tissue or organ. The transplanted cell, tissue or organ may be anallograft or xenograft.

[0052] As used herein, the terms “transplant,” “transplantation” andgrammatical variations thereof mean grafting, implanting, ortransplanting a cell, tissue or organ from one part of the body toanother part, or from one individual/animal to anotherindividual/animal. The term also includes genetically modified cells,tissue and organs, e.g., by ex vivo gene therapy in which thetransformed cells, tissue and organs are obtained or derived from theperson who then receives the transplant, or from a differentperson/animal.

[0053] Methods and compositions of the invention may be used in vitro,ex vivo or in vivo. Compositions can be administered as a single ormultiple dosage form, on consecutive or alternating days orintermittently. For example, single or multiple dosage forms can beadministered on alternating days or intermittently, over about 7 to 45days or over about 1 to 15 weeks. In one embodiment, a composition isadministered as a single dose on alternating days for between 3 and 5weeks.

[0054] Treatment usually results in an improvement in the subject'scondition, that is a change beneficial to the subject, tissue or cell orcell population in the subject that is detectable. Thus, treatment canresult in inhibiting, reducing or preventing a progression or worseningof the condition or disorder or symptoms, or further deterioration oronset of one or more additional symptoms of the condition or disorder.Thus, a successful treatment outcome leads to a “therapeutic effect,” orinhibiting, reducing or preventing the severity or frequency of symptomsor underlying causes of a disorder or condition in the subject.Stabilizing a disorder or condition is also a successful treatmentoutcome. Therefore, treatment can reduce or prevent severity orfrequency of one or more symptoms of the condition or disorder, inhibitprogression or worsening of the condition or disorder, and in someinstances, reverse the condition or disorder. Thus, in the case of animmune disorder, for example, treatment can lead to an improvement of ahistopathological change caused by or associated with the immunedisorder, for example, preventing further or reducing or regeneratingskeletal joint infiltration or tissue destruction, or thymus, kidney,liver, spleen, or skin tissue infiltration or tissue destruction.

[0055] Treatment also includes affecting the underlying causes of thecondition or disorder or symptoms thereof. Thus, in the case of animmune disorder, for example, re-regulating or normalizing absolutenumbers of lymphocytes (e.g., splenocytes) or numbers of maturelymphocytes towards normal baseline is considered a successful treatmentoutcome. Similarly, a reduction of circulating antibodies (e.g.,autoantibodies) towards normal baseline is considered a successfultreatment outcome.

[0056] The term “ameliorate” means a detectable improvement in thesubject's overall condition. A detectable improvement includes asubjective reduction in the severity or frequency of symptoms caused byor associated with the disorder or-condition, an improvement in theunderlying causes of the disorder or condition, or a reversal of thedisorder or condition, which is detectable using an assay.

[0057] Methods of the invention may be practiced prior to (i.e.prophylaxis) or after symptoms begin, before or after symptoms or thedisorder develop (e.g., before cell, tissue or organ transplantation).Administering a composition prior to or immediately followingdevelopment of symptoms may decrease the severity or frequency of thesymptoms in the subject. In addition, administering a composition priorto or immediately following development of symptoms may decrease orprevent damage to cells, tissues and organs that occurs, for example,during immune dysfunction (e.g., autoimmunity).

[0058] The term “subject” refers to animals, typically mammaliananimals, such as a non-human primate (gorillas, chimpanzees, orangutans,macaques, gibbons), a domestic animal (dogs and cats), a farm animal(horses, cows, goats, sheep, pigs), experimental animal (mouse, rat,rabbit, guinea pig) and humans. Human subjects include adults andchildren. Human subjects include those having or at risk of havingimmune dysfunction. At risk subjects can be identified through geneticscreening. Particular examples of genetically linked immune disordersthat may be identified include X-linked severe combinedimmunodeficiency, Adenosine deaminase deficiency, DiGeorge Anomaly,Ataxia-telangiectasia, Wiscott-Aldrich Syndrome, Leukocyte adhesiondeficiency, and Myotonic dystrophy. These and other disorders aredetectable through fetal blood or amniotic cells, or through adulttissue samples as described in Samter's Immunologic Diseases; M M Frank,K F Austen, H N Claman, and E R Unanue editors; Little, Brown andCompany. Reviewing family history may be used to detect inheritancepatterns or an increased risk (predisposition) of developing thedisorder (e.g., autoimmunity or immunodeficiency). At risk subjects mayalso be identified by screening for a specific characteristic, such asthe presence of undesirable or aberrant populations of lymphocytes(e.g., splenocytes) or autoantibodies. At risk subjects include those inneed of a cell, tissue or organ transplant. Subjects further includedisease model animals (e.g., such as mice and non-human primates) fortesting in vivo efficacy of the compositions of the invention (e.g.,CIA, BXSB, EAE and SCID murine models).

[0059] The invention is practiced with compounds known as “lymphocytedifferentiation factors,” which are molecules capable of regulating ormodulating cell signaling or response to signaling, which in turn canre-regulate, normalize or modulate cell behavior of the cell itself,other cells or processes in which the cells participate (e.g., immunesystem function). As set forth herein, a specific example of alymphocyte differentiation factor is PA. Lymphocyte differentiationfactors can be used in accordance with the invention in low amounts asset forth herein for PA.

[0060] The invention is also based at least in part on the low amountsof PA that can produce one or more of activities disclosed herein. Forexample, PA at 1×10⁻⁵ μg (1×10⁻¹¹ G) per dose administered onalternating days (M/W/F) starting at the time of secondary antigeninduction regulated expression and/or progression of the ensuinginflammatory response and regulated expression and/or reversed thetissue damage caused by the inflammatory response. However, at thisamount of PA, there was no substantial superantigen activity orstimulation of humoral immunity.

[0061] The invention therefore also provides compositions, including PAin an amount that is able to produce one or more of the activitiesassociated with PA, without producing substantial superantigen activity,substantial stimulation of humoral immunity or is substantiallyindependent of Fc binding. Activities of PA in such amounts include, forexample, re-regulating or normalizing aberrant or undesirable humoral orcellular immune response (modulating lymphocyte proliferation, apoptosisor differentiation), inhibiting, reversing, ameliorating or reducingautoimmunity, inflammation or an inflammatory response, or at least aportion of tissue damage caused by an undesirable or aberrant immuneresponse (inhibiting or preventing disease progression, promoting orenhancing disease reversal or tissue regeneration), and normalizing T orB splenocyte numbers or their response pattern to one or more mitogens.

[0062] Thus, in one embodiment, a composition includes PA in an amountsufficient to modulate lymphocyte proliferation, apoptosis ordifferentiation. In another embodiment, a composition includes an amountof PA in an amount sufficient to inhibit, reverse, ameliorate or reduceautoimmunity, inflammation or an inflammatory response. In yet anotherembodiment, a composition includes an amount of PA in an amountsufficient to inhibit, reverse, ameliorate or reduce at least a portionof cell, tissue or organ damage caused by an undesirable or aberrantimmune response. In still other embodiments, a composition includes anamount of PA in an amount sufficient to re-regulate or normalize T or Bsplenocyte numbers or their response to one or more mitogens. In oneaspect, the amount of PA is less than 1 μg. In another aspect, theamount of PA is less than 1 μg but greater than 0.01 picograms (pG). Inyet another aspect, the amount of PA is less than 0.5 to 0.1 μg butgreater than 0.1 pG. In still another aspect, the amount of PA is lessthan 0.1 to 0.01 μg but greater than 1 pG. In additional aspects, theamount of PA is less than 1 to 0.1 μg but greater than 1 pG; less than0.1 to 0.01 μg but greater than 1 pG; less than 0.01 to 0.001 μg butgreater than 1 pG; less than 1 to 0.5 ng but greater than 1 pG; lessthan 500 to 250 pG but greater than 1 pG; less than 250 to 50 pG butgreater than 5 pG; and less than 50 to 25 pG but greater than 5 pG,e.g., 20, 15, or about 10 pG. In yet additional aspects, the amount ofPA does not produce substantial superantigen activity, substantialstimulation of humoral immunity or is substantially independent of Fcbinding.

[0063] As used herein, the phrases “without substantial” or“substantially independent,” when used in reference to superantigenactivity, stimulation of humoral immunity or Fc binding of PA, meansthat the characteristic referred to does not contribute significantly tothe observed activity of PA at that amount. Thus, an amount of PA thatdoes not produce substantial superantigen activity means that PA'ssuperantigen activity does not contribute significantly to the activityof that amount of PA. Similarly, an amount of PA that does not producesubstantial stimulation of humoral immunity may produce a small amountof humoral activity but again the immunity produced does not contributesignificantly to PA's activity at the amount of PA used. Likewise, anamount of PA that is substantially independent of Fc binding means thatFc binding does not contribute significantly to PA's activity at theamount of PA used. In other words, removing or impairing the Fc functionof PA would not destroy PA's activity at the amount used. In general, atthe low amounts of PA used, superantigen activity, stimulation ofhumoral immunity or Fc binding of PA does not contribute significantlyto PA's activity.

[0064] Superantigen activity is typically characterized by thestimulation of non-specific subsets of T cells to proliferate. That is,T cell proliferation is largely independent of epitope specificity.Superantigen activity typically stimulates about 5-10% of T cells toproliferate whereas a conventional antigen may stimulate about 1 in 10⁶cells in an individual. Superantigen activity may therefore be assayedby determining numbers of T cells that are stimulated to proliferate.Examples of such assays are described, for example, in Johnson et al.,Scientific American, April 1992. pp. 92-101; and Kotzin et al., Adv.Immunol. 54:99 (1993). Superantigen and FC binding assays are described,for example, in Romagnani et al., J. Immunol. 129:596 (1982). FC bindingassays are described, for example, in Langone J J, Adv. Immunol. 32:157(1982). Stimulation of humoral immunity assays are described, forexample, in Leonetti et al., J. Exp. Med. 189:1217 (1999).

[0065] Methods of the invention can therefore be practiced using thecompositions of the invention. For example, in one embodiment, aneffective amount of PA is a dose of about 0.1 picogram to about 1microgram. In another embodiment, an effective amount of PA is a dose ofabout 1 picogram to about 1 microgram. In yet another embodiment, aneffective amount of PA is a dose of about 10 picograms to about 1microgram. In still another embodiment, an effective amount of PA is adose of about 10 picograms to about 0.1 microgram. In additionalembodiments, an effective amount of PA is a single dose of about 10picograms to about 0.1 microgram.

[0066] Compositions may be administered systemically or locally by anyroute. For example, PA may be administered intravenously, orally (e.g.,ingestion or inhalation), intramuscularly, intraperitoneally,intradermally, subcutaneously, intracavity, intracranial, transdermally(topical), parenterally, e.g transmucosal and rectally. Compositions ofthe invention including pharmaceutical formulations can be administeredvia a microencapsulated delivery system or packaged into an implants foradministration.

[0067] Compositions further include pharmaceutical formulationscontaining PA in an amount having one or more of the activitiesdisclosed herein. In various embodiments, a pharmaceutical formulationincludes PA in an amount sufficient to re-regulate or normalize aberrantor undesirable humoral or cellular immune response (modulatinglymphocyte proliferation, apoptosis or differentiation), inhibit,reverse, ameliorate or reduce autoimmunity, inflammation or aninflammatory response, or at least a portion of tissue damage caused byan undesirable or aberrant immune response (inhibiting or preventingdisease progression, promoting or enhancing disease reversal or tissueregeneration), normalize T or B splenocyte numbers or their response toone or more mitogens, without substantial superantigen activity, withoutsubstantial stimulation of humoral immunity or substantially independentof Fc binding, and a pharmaceutically acceptable carrier or excipient.

[0068] As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, excipients, diluents andthe like that can be administered to a subject, preferably withoutproducing excessive adverse side-effects (e.g., nausea, abdominal pain,headaches, etc.). Such preparations for administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions.

[0069] Pharmaceutical formulations can be made from carriers, diluents,excipients, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with administration to a subject. Such formulations canbe contained in a tablet (coated or uncoated), capsule (hard or soft),microbead, emulsion, powder, granule, crystal, suspension, syrup orelixir. Supplementary active compounds and preservatives, among otheradditives, may also be present, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.

[0070] A pharmaceutical formulation can be formulated to be compatiblewith its intended route of administration. Thus, pharmaceuticalformulations include carriers, diluents, or excipients suitable foradministration by routes including intraperitoneal, intradermal,subcutaneous, oral (e.g., ingestion or inhalation), intravenous,intracavity, intracranial, transdermal (topical), parenteral, e.g.transmucosal and rectal.

[0071] Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following: a sterile diluentsuch as water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

[0072] Pharmaceutical formulations suitable for injection includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. Fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride can beincluded in the composition. Prolonged absorption of injectableformulations can be achieved by including an agent that delaysabsorption, for example, aluminum monostearate or gelatin.

[0073] For oral administration, a composition can be incorporated withexcipients in the form of tablets, troches, or capsules, e.g., gelatincapsules. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included in oral formulations. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or flavoring.

[0074] Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including materials that slowly degradewithin the body and in turn release the active ingredient(s). Forexample, a time delay material such as glyceryl monostearate or glycerylstearate alone, or in combination with a wax, may be employed.

[0075] Additional formulations include biodegradable or biocompatibleparticles or a polymeric substance such as polyesters, polyamine acids,hydrogel, polyvinyl pyrrolidone, polyanhydrides, polyglycolic acid,ethylene-vinylacetate, methylcellulose, carboxymethylcellulose,protamine sulfate, or lactide/glycolide copolymers,polylactide/glycolide copolymers, or ethylenevinylacetate copolymers inorder to control delivery of an administered composition. Methods forpreparation of such formulations will be apparent to those skilled inthe art. The materials can also be obtained commercially from AlzaCorporation and Nova Pharmaceuticals, Inc., for example.

[0076] The rate of release of a composition can be controlled byaltering the concentration or composition of such macromolecules. Forexample, the composition can be entrapped in microcapsules prepared bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0077] Additional pharmaceutical formulations appropriate foradministration are known in the art and are applicable in the methodsand compositions of the invention (see, e.g., Remington's PharmaceuticalSciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The MerckIndex (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; andPharmaceutical Principles of Solid Dosage Forms, Technonic PublishingCo., Inc., Lancaster, Pa., (1993)).

[0078] Compositions of the invention can include combinations of othercompositions, and be included in the pharmaceutical compositions of theinvention. For example, a drug that reduces an inflammatory response orinflammation or that stimulates differentiation of a cell can beincluded with a low amount of PA. Exemplary drugs include steroidal(SAI) and non-steroidal anti-inflammatory's (NSAI), for example, acorticosteroid, a cox-2 inhibitor, or drugs that affect the immunesystem such as chemokines and cytokines such as interleukins andinterferons.

[0079] Compositions of the invention, including pharmaceuticalformulations can be packaged into kits, which optionally can containinstructions for use, for example, practicing a method of the invention.The invention therefore provides kits. In one embodiment, a kit includesone or more compositions of the invention (e.g., PA), includingpharmaceutical formulations, packaged into suitable packaging material.In additional embodiments, a kit includes a label or packaging insertfor practicing a method of the invention. Thus, in one embodiment, a kitincludes instructions for treating a subject having or at risk of havingan immune disorder or dysfunction, in vitro, in vivo, or ex vivo. In yetadditional embodiments, a kit includes a label or packaging insertincluding instructions for treating a subject having an autoimmunedisorder with low amounts of PA in vivo, or ex vivo.

[0080] As used herein, the term “packaging material” refers to aphysical structure housing the components of the kit. The packagingmaterial can maintain the components sterilely, and can be made ofmaterial commonly used for such purposes (e.g., paper, corrugated fiber,glass, plastic, foil, ampules, etc.). The label or packaging insert caninclude appropriate written instructions, for example, practicing amethod of the invention. Kits of the invention therefore canadditionally include instructions for using the kit components in amethod of the invention.

[0081] Instructions can include instructions for practicing any of themethods of the invention described herein. Thus, inventionpharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration to a subject.Instructions may additionally include indications, a satisfactoryclinical endpoint, any adverse symptoms that may occur, or additionalinformation required by the Food and Drug Administration for use on ahuman subject.

[0082] The instructions may be on “printed matter,” e.g., on paper orcardboard within the kit, on a label affixed to the kit or packagingmaterial, or attached to a vial or tube containing a component of thekit. Instructions may comprise voice or video tape which can optionallybe included on a computer readable medium, such as a disk (floppydiskette or hard disk), optical CD such as CD- or DVD-ROM/RAM, magnetictape, electrical storage media such as RAM and ROM and hybrids of thesesuch as magnetic/optical storage media.

[0083] Invention kits can also include one or more drugs that provide asynergistic or additive effect or that reduce or ameliorate one or moresymptoms of a drug or disorder. For example, a drug that reduces aninflammatory response or inflammation may be included. Exemplary drugsinclude steroidal (SAI) and non-steroidal anti-inflammatory's (NSAI),for example, a corticosteroid, or a cox-2 inhibitor. Invention kits canadditionally include a buffering agent, a preservative, or a stabilizingagent. The kit can further include control components for assaying anactivity or effect of treatment. Each component of the kit can beenclosed within a separate individual container. For example, a kit caninclude a single unit dosage of a low amount of PA as set forth herein(e.g., from less than 1 μg to 1 pG). Alternatively, a kit can includemultiple unit dosage forms of a low amount of PA. For example, each ofthe multiple unit dosage forms would contain a low amount of PA in aseparate individual container (e.g., each unit dose of PA would be fromless than 1 μg to 1 pG per dose). Kit components can be in a mixture ofone or more containers and all of the various containers can be withinsingle or multiple packages.

[0084] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described herein.

[0085] All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

[0086] As used herein, the singular forms “a”, “and,” and “the” includeplural referents unless the context clearly indicates otherwise. Thus,for example, reference to “a lymphocyte” includes a plurality of suchcells.

[0087] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLE 1

[0088] This example describes an animal inflammation (arthritis) modeland histological data indicating that PA administered at very lowconcentrations can reduce inflammation and inhibit or reverse tissuedamage caused by inflammation.

[0089] Three separate studies with three groups of five animals each (atotal of 15 animals per treatment group). The first group is control,injected with phosphate buffered saline) PBS carrier. The second groupreceived 100 μg Enbrel per mouse per day. This was the optimal Enbreldose as described by the manufacturer (Immunex, Corp., Seattle, Wash.).The third group was injected with 10 picograms (pG) of PA in PBS carrieron Monday, Wednesday, and Friday during the treatment period(Amersham/Pharmacia Biotech, Piscataway, N.J.). PA may also be obtainedfrom Sigma-Aldrich, St. Louis, Mo.; Pierce Chemical Co., Pittsburgh,Pa.; and Calbiochem, San Diego, Calif..

[0090] Table 1 below summarizes the clinical index data for the controlgroup indicating a progressive inflammatory response in thesesusceptible animals. The response does not peak or plateau within thetime limits used and control animals were sacrificed when the responsejeopardized their health status. TABLE 1 Control Clinical IndexReading-CIA Model Mean S.D. SEM N Day 0 0 0 0 0 0 0 0 30 1 0.00 0.000.00 5.00 2 0.00 0.00 0.00 25.00 3 0.25 0.50 0.25 4.00 4 0.08 0.28 0.0625.00 5 1.00 0.94 0.30 10.00 7 0.96 1.06 0.21 25.00 8 1.60 1.51 0.4810.00 9 1.40 1.35 0.30 20.00 10 3.52 3.44 0.69 25.00 15 7.80 5.63 2.525.00 16 10.80 3.11 1.39 5.00 18 2.25 1.59 0.35 20.00 19 2.58 1.50 0.3419.00 23

[0091] Table 2 shows similar data for the caliper measurements of thepaws of the control group animals during the same time period. Thesedata mirror the clinical index data with the exception that a plateau inthe response appears after day 10. This response plateau correspondswith the kinetics of typical antibody induced inflammatory responses.TABLE 2 Control Paw Measurement-CIA Model Mean S.D. SEM N 165.80 9.312.08 20.00 0 164.95 11.91 1.52 61.00 1 168.50 6.13 1.37 20.00 2 168.508.90 1.41 40.00 3 172.30 4.75 1.06 20.00 4 171.38 7.26 1.15 40.00 5167.00 16.12 2.55 40.00 7 182.35 19.06 3.01 40.00 8 176.44 19.56 3.0541.00 9 207.45 36.93 8.26 20.00 10 212.28 38.03 6.01 40.00 15 204.3031.25 6.99 20.00 16 212.95 36.18 8.09 20.00 18 217.40 44.90 10.04 20.0019 224.30 44.01 9.84 20.00 23

[0092] Histological analysis of knee and ankle of a control animal takenon day 15 of the inflammatory response indicated extensive immuneinfiltration and tissue destruction. Immunocytes accumulated in thesynovium of control animal. Enbrel treated animals at day 35 showedcontinued immune infiltration in the synovium of knee and ankle andevidence of continued tissue destruction.

[0093] Table 3 summarizes the histological examination results of theknee and ankle joints of control, untreated DBA/1 animals. These ratingsare assigned on a blinded basis by the histologist on a continuous scalefrom 1 to 10 with 1 representing a “normal” histological appearance and10 a high degree of damage. Knee and ankle joints of the control animalsafter collagen induction show maximal tissue damage (“10”s). Thiscorrelates with both the Clinical index (Table 1) and physicalmeasurement data (Table 2). TABLE 3 Histology Rating of CIAModel-Control Animals Histology Tissue Rating N Ankle 10 15 Knee 10 15

[0094] PA treated animals at day 35 of the inflammatory response showedmuch less evidence of immune infiltration and only slight evidence oftissue destruction. Only a few host immunocytes were present in thesynovium and there was no evidence of tissue fragments or tissuedestruction. These data therefore demonstrate that PA reduces acuteinflammation in the CIA model. These data also demonstrate that PAreduces tissue damage or promotes tissue repair more than Enbrel.

[0095] Tables 4 and 5 show the results of 3 separate studies testing theeffect of PA (10 pG/injection, M/W/F). Enbrel was tested for comparison;the results obtained were comparable with published results. PAtreatment was very effective, reaching significance at approximately 10%of the number of animals required for the Enbrel standard. The resultsfor the total clinical index (Table 4) and the physical measurements(Table 5) are comparable. TABLE 4 PA Treatment on the CIA model (TotalClinical Index) Pooled Data: C.I. Treatment Day Mean N P[1-tail] Control15 3.52 25 17 3.88 25 19 2.25 20 Protein-A 15 1.92 25 0.03 17 1.95 240.02 19 1.40 20 0.04 Enbrel 15 2.79 24 0.17 17 3.41 24 0.30 19 2.85 200.11

[0096] TABLE 5 PA Treatment on the CIA model (Measurement) Pooled Data:Meas. Treatment Day Mean N P[1-tail] Control 15 212.3 40 17 218.1 40 19217.4 20 Protein-A 15 195.4 36 0.02 17 191.4 36 0.001 19 197.2 20 0.05Enbrel 15 204.2 36 0.18 17 209.1 36 0.18 19 230.3 20 0.19

[0097] Table 6 shows the results of a histological assessment of DBA/1mice sacrificed at 1, 2 and 3 weeks during their treatment regimen. BothPA and Enbrel treatments show significant damage at the tissue level.These data demonstrate that in spite of the significant decreases inTotal clinical index and paw measurements (Tables 4 and 5) there isstill damage at the tissue level. PA treatment appears to delay tissuedamage (treatment week one versus control) but does not prevent thedamage. TABLE 6 PA treatment and histological damage Control PA Enbrel 1wk Ankle: 10 Ankle: 8 Ankle: 1-5 Knee: 10 Knee: 0-5 Knee: 0-10 2 wkAnkle: 10 Ankle: 10 Ankle: 10 Knee: 10 Knee: 10 Knee: 10 3 wk Ankle: 10Ankle: 10 Ankle: 10 Knee: 10 Knee: 10 Knee: 10

[0098] DBA/1 mice induced with Type II Collagen as per the CIA modelprotocol were treated immediately after the second antigeninjection—solvent carrier (PBS) for the control, PA (at 10 pG perinjection M/W/F), and Enbrel (100 ug/injection every day).

[0099] Table 7 shows histological results after extending treatment to35 days. Control mice were sacrificed at 18 days for humanitarianreasons and their 18 day data are included for comparison. Enbreltreatment showed no amelioration of the histological damage. Incontrast, PA treatment reversed histological damage at 14-21 days. Thishistological data correlates with the Total Clinical Index of theseanimals. Thus, PA treatment significantly reduced the severity of theacute inflammatory response and continuing treatment reversed theexisting tissue damage caused by the response. TABLE 7 PA extendedTreatment and Histological assessment Control @ PA @ 35 Enbrel 18 DaysDays @ 35 Days rating Ankle: 10 Ankle: 0-1 Ankle: 10 Knee: 10 Knee: 0-1Knee: 10

[0100] The histological assessment of these tissues included low,medium, and high magnification assessment. In both control (at 18 days,time of sacrifice) and Enbrel treatment groups the synovium had largenumbers of activated lymphocytes, whereas the PA group at day 35 oftreatment had few small lymphoid cells which were similar in number andmorphology to those in the DBA/1 animals prior to Type II collagenantigen induction.

[0101] In sum, these data demonstrate that paw measurements and theclinical index assessments document the induced inflammatory response inthe CIA animal model; that PA reduces the inflammatory response duringthe acute phase(P values vs control <0.05) and reverses the histologicaldamage caused by the response by day 35 of treatment; that PA has itsameliorative effect at concentrations and dosing schedules predicted bythe BXSB animal model and Tissue Culture assessments (discussed furtherbelow); and that Enbrel does reduce inflammation on Day 14 (notsignificantly with N=15) but does not reduce inflammatory damageobserved at day 35, indicating that PA is more effective than Enbrel.

EXAMPLE 2

[0102] This example describes data indicating that the mechanism ofaction (MOA) of PA appears distinct from Enbrel.

[0103] The accepted MOA for the CIA animal model is competitiveinhibition of the expression of α-TNF. Enbrel is a known α-TNFinhibitor. Regression analysis of the Enbrel data indicated a delay inthe onset of the inflammatory response. In contrast, regression analysisof the PA data suggested an alteration in the inflammatory processitself. The MOA of PA therefore does not appear to be primarily throughα-TNF inhibition. In addition, PA is not only more effective than Enbrelin reducing the induced inflammatory response in treated animals but isalso capable of reversing pre-existing tissue damage caused by thatresponse.

[0104] While not being bound by any theory, PA may therefore “modulate”a basal control mechanism responsible for integrating immune-dependentresponses. This MOA would encompass α-TNF inhibition but from aself-regulatory perspective instead of simple target moleculecompetitive inhibition. Such a MOA is predicted to have the followingproperties:

[0105] 1) small amounts required—a regulatory effect on a primitivecontrol mechanism that “branches” to influence additional mechanisms;

[0106] 2) self-regulatory—if PA acts at an early control point then thesystem will have the ability to regulate the intensity and direction ofthe mechanism resulting in few if any side effects;

[0107] 3) pleiotropic target i.e. non-cell-lineage specific—if PAcontrol point is early then the subsequent control should be diverse,and the concentration of PA and dosing schedule should be constant; and

[0108] 4) PA effector molecule structure should be found in associationwith a number of more complex structures.

EXAMPLE 3

[0109] This example describes data indicating that PA has multiplemodulatory activities in an animal model characterized by a combinedautoimmune deficiency disease having a genetic basis resulting in earlydeath. In particular, PA prevents early onset of wasting, expansion ofthe splenic compartment, regulates humoral immunity (autoantibodies),cellular immunity (TH₁/TH₂ balance) and lymphoid cell differentiation aswell as ameliorating tissue damage caused by the disease processes.

[0110] The BXSB murine model is a gene (Yaa) based animal model thatmanifests with early death in males, typically from kidney failure. Thismodel is considered in the literature as an analog for human systemiclupus. The gene defect expresses as a series of inter-relatedprogressive systemic autoimmune diseases having the following pattern:thymic atrophy, anti-nuclear antibody, liver disease, arthritic disease,kidney disease and early death.

[0111] Because this is a genetic model with multiple outcomes previousstudies by other investigators have concentrated on single aspects ofthe disease. The effect of PA on multiple aspects of the disease processstudied herein include:

[0112] 1. overall effect on the animal's physiology

[0113] a. growth curves

[0114] b. histology of thymus, liver, brain, kidney, ankle, and knee

[0115] 2. immune regulation—cellular proliferation/apoptosis

[0116] a. splenic size, and

[0117] b. cell count

[0118] 3. lymphocyte dynamics

[0119] a. T/B responses to mitogenic stimuli

[0120] 4. lymphocyte function

[0121] a. Humoral immunity

[0122] i. Ig-PFC production

[0123] ii. Auto-antibody: ANA, anticardiolipin

[0124] b. Cellular Immnuity

[0125] i. Natural killer

[0126] c. Cell surface markers

[0127] d. Cellular cytokines

[0128] Study Design:

[0129] 1. Chronic Treatment, Abnormal: groups of 25 male BXSB (control+4treatment groups) were treated with PA on M/W/F over a 15 week periodwith periodic peel-off sacrifices (usually every 3 weeks of treatment).

[0130] 2. Chronic Treatment, Normal: groups of 15 male C57BL/6J weretreated with PA on M/W/F over a 15 week period with periodic peel-offsacrifices (usually every 3 weeks of treatment).

[0131] 3. Acute Treatment, Abnormal: groups of 15 male BXSB were treatedfor 3 weeks with amounts of PA determined above, and then animals weresacrificed 3,6 and 9 weeks post treatment.

[0132] 4. Acute Treatment, Normal: groups of 15 male C57BL/6J weretreated for 3 weeks with amounts of PA determined above, and thenanimals were sacrificed 3,6 and 9 weeks post treatment.

[0133] Determination of PA Concentration:

[0134] PA amounts were administered to BXSB over eight logs ofconcentration, from 1 μG/injection to 10⁻⁷ μG/injection. The resultsindicate two PA optima, one at 0.01 μG/injection and another at 10⁻⁵μG/injection. The shape of the dose-response curves is Gaussian. For thesake of clarity the data presented is for 10⁻⁵ μG/injection (FIG. 1).

[0135]FIG. 1 shows the weight gain of BXSB males following PAadministration. The weights were taken each time the animals wereinjected with either carrier or PA. Panel A is the weight gain growthcurve for a normal C57BL/6J mouse strain, and is presented to show thetypical shape of a normal weight gain growth curve. Panel B shows thecumulative sum of weight gain data from 25 control BXSB male mice. Thiscurve shows a weight peak at approximately 4 months of age followed by adecrease in body weight, which corresponds to the reported onset of theBSXB autoimmune disease. The decrease in body weight leads to the“wasting” syndrome linked to immune complex deposition in the kidney.

[0136] Panel C shows the effect of chronic administration of PA at theoptimal two concentrations (8 concentrations studied). Both the 0.01μG/injection and 0.00001 μG/injection show significant changes in boththe shape of the weight gain growth curve comparing better with thenormal logistic shape than with the BXSB control and the average weightof both the treated animal groups is significantly higher than control(X=24.16 P=0.0002, for the 10⁻⁵ μG dose).

[0137] In order to analyze these growth curves and compare them whentreating groups of BXSB animals with PA a regression analysis wasperformed on the data. The C57BL/6J growth data presented above, is bestrepresented by the following cubic equation:

C57BL/6J Control—y=−0.0286x³+0.4067x²−0.8452x+16.816[R²=0.9886]

[0138] The R-squared value indicates an extremely good fit between theactual data and the line equation. The equation itself is a relativelysimple extraction of a “logistic” shaped curve, which is typical ofnormal growth curves (FIG. 1).

[0139] There are four separate data sets for the control BXSB growthcurve. All data sets agree internally and the overall equation for thepooled data is as follows:

Pooled BXSB−y=−0.0001x³+0.0034x²+0.1851x+18.746 [R²=0.9384]

[0140] This equation is a quantitative representation of the differencesin curve shape noted between FIG. 1, panels A and B. The cubic andquadratic function define the overall shape and the very low valuesdefine the rate of increase in weight and the “wasting” phase where theweights are seen to decrease as a function of time. In this study theaim is not to make the BXSB growth curve “look like” the C57BL/6J curve,as each strain has there own specific growth characteristics, but ratherto increase the rate of growth initially and decrease or eliminate the“wasting phase” evident in FIG. 1, panel D.

[0141] Table 8 shows the fitted equations for the various PA treatmentgroups of BXSB animals. The slope of the initial growth curve, whichindicates growth rate, is severely restricted in BXSB control animals.The inflexion point in BXSB control animals indicates the lethal effectof the disease process. The results indicate that PA treatment has apositive effect on both growth rate and lethality in a dose dependentmanner. TABLE 8 PA effect on the growth kinetics of BXSB TreatmentGrowth equation Pooled BXSB y = −0.0001x3 + 0.0034x2 + 0.1851x + 18.746R2 = 0.9384 Rx 1.0 PA y = 4E−06x3 − 0.4942x2 + 18171x − 2E+08 R2 =0.9087 Rx 0.1 PA y = −1E−04x3 + 10.697x2 − 392306x + 5E+09 R2 = 0.9463Rx 0.01 PA y = 3E−05x3 − 2.8056x2 + 102980x − 1E+09 R2 = 0.918 Rx 0.001PA y = 1E−05x3 − 1.3946x2 + 51161x − 6E+08 R2 = 0.9773 Rx 0.0001 PA y =−4E−05x3 + 4.2053x2 − 154454x + 2E+09 R2 = 0.9487 Rx 0.00001 y =−1E−05x3 + 1.4883x2 − 54653x + 7E+08 PA R2 = 0.8665 Rx 0.000001 y =3E−05x3 − 2.9251x2 + 107455x − 1E+09 PA R2 = 0.8682 Rx y = −5E−06x3 +0.5628x2 − 20666x + 3E+08 0.0000001 R2 = 0.9474 PA

[0142] Histological Analysis:

[0143] The histological deterioration of various organs of the BXSBmouse model is the hallmark of a combined autoimmune deficiency disease.This syndrome is multi-facetted and involves a number of organs. Theorgan damage is progressive and individual damage may vary according totissue type.

[0144] Table 9 shows the results from the histological analysis ofhigher amounts of PA (1 to 0.001 μg/injection) and indicates that PAchanged the onset and/or severity of the histological changes in a dosedependent manner. Table 10 shows similar data for lower amounts of PA(0.00001 to 0.0000001 μg/injection). Both PA doses exhibited thegreatest improvement in organ histology. Tables 9 and 10: Histology ofBXSB BXSB Rx 0.01 μg Rx 0.001 μg Tissue Control Rx 1 μg PA Rx 0.1 μg PAPA PA Thymus 4+ 4+ 4+ 4+ 4+ (10-20 wks) (16-20 wks) (10-20 wks) (16-20wks) (12-13 wks) Liver local N/A local local local (double inflammationinflammation inflammation inflammation nuclei) 20 wks 19 wks 10 wks 20wks Kidney normal normal normal normal normal Knee normal normal normalinflammation normal 16 wks Ankle fibers inflammation inflammationinflammation normal disintegrating 19 wks 10 wks 16 wks 20 wks BXSB Rx0.0001 μg Rx 0.00001 μg Rx 0.000001 Rx 0.0000001 Tissue Control PA PA μgPA μg PA Thymus 4+ normal 4+ 4+ 4+ (11-22 wks) (11-22 wks) (22 wks) (22wks) Liver local normal local normal local (double inflammationinflammation inflammation nuclei) 20 wks 19 wks 11 wks Kidney normalnormal normal normal normal Knee normal normal normal normal normalAnkle normal normal normal normal normal

[0145] The results in Tables 9 and 10 indicate that PA treatment of BXSBmice was able to decrease autoimmune damage in a dose dependent fashion.Treatment delayed the onset of thymic atrophy, and reduced the severityof liver and joint inflammation. The kidneys remained with in normalrange. The tissues of PA treated animals exhibited a reversal of thedamage present in BXSB control tissues, although they were notcompletely normal as compared to C57BL/6J animals. The optimal PA dosewas 0.0001 μg, which is consistent with other assay systems (e.g.,Ig-PFC production, NK activity, mitogen response pattern, cytokineproduction, autoantibody production, spleen size, histologicalimprovements).

[0146] In sum, the studies indicate that PA prevents the early onset ofwasting, and ameliorates or reverses damage caused by autoimmune diseaseprocesses in tissues including thymus, liver, kidney, knee and ankle.

EXAMPLE 4

[0147] This example describes data indicating that PA has immunemodulatory activity (e.g., proliferation, apoptosis or differentiation)in spleen reflected by inhibition of spleenic expansion and splenocytecell numbers.

[0148] Spleens were removed from animals during the studies. NormalC57BL/6J spleen was a reference point. Spleens from untreated BXSBanimals were significantly enlarged. In contrast, PA treated BXSB animalspleens resembled the size of normal C57BL/6J spleen.

[0149] The enlarged spleens in untreated BXSB animals is known assplenomegalia, and is the result of either a massive systemic infectionprocess or of aberrant cellular apoptosis or proliferation. Becausethese animals are maintained in a sterile environment the enlargedspleen is likely the result of faulty cell growth/death control. Anotherexplanation for enlarged spleen would be that the cells had increased intheir individual size, a process known as blastosis. Examination of thefluorescently activated cell sorting (FACS) data to measure cell sizeruled out this possibility. FACS analysis revealed that although a trendto slightly larger cells was observed in the BXSB controls, nostatistically significant increase in cell size was observed. Consistentwith these findings is that BXSB splenocyte numbers are more thanfive-times those of normal animals (Table 11, top). TABLE 11 PA Effecton Splenomegalia BXSB control C57BL/6J DBA/2J MEAN 5.E+08 9.E+07 1.E+08SEM 4.E+07 7.E+06 6.E+06 N 20 20 20 Rx 1.0 PA P-value Rx 0.1 PA P-valueRx 0.01 PA P-value Rx 0.001 PA P-value MEAN 1.E+08 8.68E−03 1.E+087.32E−04 7.E+07 1.02E−06 1.E+08 8.92E−06 SEM 7.E+07 5.E+07 4.E+07 4.E+07N 3 3 5 4 Rx E−04 PA P-value Rx E−05 PA P-value Rx E−06 PA P-value RxE−07 PA P-value MEAN 3.E+08 1.73E−02 1.E+08 6.70E−05 3.E+08 9.54E−022.E+08 7.26E−05 SEM 6.E+07 3.E+07 1.E+08 4.E+07 N 4 3 4 5

[0150] Table 11 (top) shows the contents of the spleens of both controlmouse strains, C57BL/6J and DBA/2J, and BXSB males. BXSB splenocyte cellnumbers are more than 5 times those found for either of the normalstrains. PA treatment significantly reduced BXSB splenocyte numbers(Table 11, bottom).

[0151] In sum, the studies indicate that PA regulates expansion of thespleenic compartment (proliferation/apoptosis) caused by the autoimmunedisease and splenocyte numbers. PA treatment both reduced spleen sizeand splenocyte numbers significantly in a dose dependent fashion.

EXAMPLE 5

[0152] This example describes data indicating that BXSB animals exhibitaberrant splenocyte differentiation or proliferation or apoptosis. Thisdata therefore indicates that PA activity in BXSB spleen includesre-regulating aberrant/deficient splenocyte differentiation orsplenocyte proliferation/apoptosis (i.e. restoring normal cellproliferation or apoptosis).

[0153] Mitogens are lectins that have the non-specific capacity tostimulate cellular division in general populations of cells (e.g., Tand/or B lymphocytes). Lectins are found in plants and animals and arebest characterized as precursors to modern day antibody molecules.Lectins are used for intracellular and other forms of communication.

[0154] To determine whether the T-lymphocyte compartment in BXSB mice isaberrant, two T-cell specific and two B-cell specific mitogens were usedto study the response of splenocytes isolated from BXSB mice. 7different mitogen concentrations and 4 kinetic time points were used totest BXSB animals' splenocyte response in terms of the responseamplitude (an indication of the number of cells present), and secondly,the optimal stimulation concentration (an indication of thedifferentiation status of the cells).

[0155] In brief, BXSB and control mice were sacrificed periodicallyduring the treatment regimen and their spleens removed. Splenocyte cellsuspensions at 1 to 2×10⁶/ml are dispensed into 96 well plates. Mitogens(10 μG/10 μL to 0.01 μG/10 μL) were added in triplicate to the wells andthe cultures harvested at 24 hour intervals from 24 to 96 hours ofculture. All cultures were treated with tritiated thymidine for 16 hoursprior to harvest. The DNA, including the newly synthesized radiolabeledDNA, is extracted on glass fiber filters and the radioactivitydetermined by liquid scintillation counting. TABLE 12 Normal (C57BL/6)mitogen responses (Stimulation Index, S.I.) PHA: SI 10.00 5.00 1.00 0.500.10 0.05 0.01 MEAN 24.56 29.54 33.39 19.95 3.07 1.79 1.03 S.D. 11.3913.47 14.00 11.47 3.40 1.69 0.57 SEM 2.15 2.55 2.65 2.17 0.64 0.32 0.11Count 27 27 27 27 27 27 27 Con-A: SI 10.00 5.00 1.00 0.50 0.10 0.05 0.01MEAN 0.57 1.69 48.07 11.79 1.83 2.00 2.39 S.D. 0.27 1.50 32.63 15.801.94 1.08 2.99 SEM 0.05 0.28 6.17 2.99 0.37 0.20 0.56 Count 27 27 27 2727 27 27 SEB: SI 10.00 5.00 1.00 0.50 0.10 0.05 0.01 MEAN 18.70 15.7910.61 7.87 5.23 3.41 1.47 S.D. 11.49 8.69 6.14 5.57 4.57 2.47 0.55 SEM2.17 1.64 1.16 1.05 0.86 0.47 0.10 Count 27 27 27 27 27 27 27 LPS: SI10.00 5.00 1.00 0.50 0.10 0.05 0.01 MEAN 105.66 103.65 94.60 85.92 69.9559.05 39.44 S.D. 46.55 46.89 46.30 52.67 41.29 36.17 29.92 SEM 8.80 8.868.75 9.95 7.80 6.84 5.66 Count 27.00 27.00 27.00 27.00 27.00 27.00 27.00

[0156] Table 12 shows the mitogen responses of C57BL/6J mice tophytohemaglutinin (PHA), concanavalin A (Con-A), staphylococalenterotoxin B (SEB), and lipopolysaccharide (LPS). PHA and Con-Astimulate basic T lymphocyte populations to proliferate in a dosedependent fashion, whereas SEB, and LPS stimulate the dose dependentproliferation of B-lymphocyte populations. TABLE 13 BXSB mitogenresponses PHA: SI 10.00 5.00 1.00 0.50 0.10 0.05 0.01 Mean 4.63 2.173.64 4.29 3.77 2.55 1.95 S.D. 6.29 1.40 1.56 2.11 1.76 0.81 0.88 SEM0.87 0.19 0.22 0.29 0.24 0.11 0.12 N 39 39 39 39 39 39 39 Con-A: SI10.00 5.00 1.00 0.50 0.10 0.05 0.01 Mean 0.99 2.42 6.34 7.08 3.93 1.881.67 S.D. 1.02 2.07 4.32 3.69 3.46 0.97 0.93 SEM 0.14 0.29 0.60 0.510.48 0.14 0.13 N 39 39 39 39 39 39 39 SEB: SI 10.00 5.00 1.00 0.50 0.100.05 0.01 Mean 6.05 6.10 5.21 4.70 3.00 2.53 1.91 S.D. 3.77 3.18 2.693.48 1.66 1.85 1.04 SEM 0.52 0.44 0.37 0.48 0.23 0.26 0.14 n 39 39 39 3939 39 39 LPS: SI 10.00 5.00 1.00 0.50 0.10 0.05 0.01 Mean 26.40 27.7629.58 24.83 10.85 7.08 3.97 S.D. 18.99 20.58 20.78 14.66 5.50 3.76 1.64SEM 2.63 2.85 2.88 2.03 0.76 0.52 0.23 N 39 39 39 39 39 39 39

[0157] Table 13 shows the results from the mitogen study of BXSBcontrol. Statistical analyses indicates that all mitogen responses aresignificantly lower than C57BL/6J control. In addition, the shape of thePHA and Con-A response curves for BXSB control are different from normalC57BL/6J control. For both there was a shift to lower mitogenconcentrations to achieve a peak response. These changes in responsecurve shape have been associated with changes in differentiation status.TABLE 14 Statistical Comparison of BXSB Mitogen Responses to Control10.00 5.00 1.00 0.50 0.10 0.05 0.01 PHA 1.E−10 2.E−11 2.E−11 5.E−075.E−02 N.S. N.S. Con-A N.S. N.S. 6.E−07 6.E−02 N.S. N.S. N.S. SEB 2.E−054.E−06 4.E−05 2.E−04 6.E−04 1.E−03 N.S. LPS 5.E−09 2.E−08 5.E−07 5.E−068.E−08 8.E−08 N.S.

[0158] Table 14 shows a statistical comparison of control BXSB. Mitogenresponses to control C57BL/6. In almost every case, the amplitude of themitogen response in BXSB is suppressed indicating a dimunition of themature cell population and a shift in the optimal response concentrationto a lower value. These data therefore indicate aberrant differentiationin control BXSB splenocytes which leads to over-proliferation ordecreased apoptosis of splenocytes. TABLE 15 PHA Response as a Functionof PA treatment PHA: S.I. 10.00 5.00 1.00 0.50 0.10 0.05 0.01 1 PA 2.233.27 8.64 11.52 10.91 4.63 1.86 0.1 PA 1.08 1.82 4.13 5.25 4.76 2.952.62 0.001 PA 1.52 1.21 2.70 4.41 3.90 2.57 1.94 0.0001 5.53 4.03 9.6515.41 10.91 3.91 1.95 PA 1E−04 PA 5.71 2.98 5.14 6.82 6.22 2.73 1.661E−05 PA 7.73 7.00 8.34 9.80 7.26 3.47 1.89 1E−06 PA 2.70 3.13 4.47 4.823.84 3.29 2.20 1E−07 PA 5.08 5.84 3.41 4.22 3.90 2.37 2.00

[0159] TABLE 16 Con-A Response as a Function of PA treatment Con-A: S.I.10.00 5.00 1.00 0.50 0.10 0.05 0.01 1 PA 1.42 4.88 23.61 36.83 6.57 2.702.07 0.1 PA 1.29 5.43 8.51 8.06 2.00 2.20 1.64 0.001 PA 1.34 3.31 8.2617.4 9.14 3.33 1.92 0.0001 1.92 2.05 27.27 51.59 10.40 2.10 2.02 PA1E−04 PA 0.56 1.31 7.29 15.26 6.87 4.56 1.79 1E−05 PA 0.76 3.10 5.3114.29 7.69 2.25 1.34 1E−06 PA 1.01 2.34 5.04 15.98 8.03 2.85 2.52 1E−07PA 1.65 2.90 9.05 22.83 8.10 2.17 2.05

[0160] TABLE 17 SEB Response as a Function of PA treatment SEB: S.I.10.00 5.00 1.00 0.50 0.10 0.05 0.01 1 PA 13.39 11.88 12.41 8.39 6.424.73 3.76 0.1 PA 6.25 6.57 4.29 4.15 2.37 1.68 1.32 0.001 PA 7.51 7.5410.06 7.37 5.76 5.28 3.82 0.0001 PA 14.60 12.60 9.55 5.72 4.66 4.21 2.051E−04 PA 8.01 5.15 6.48 4.55 5.12 2.97 1.34 1E−05 PA 6.92 8.02 7.94 5.284.13 3.54 3.40 1E−06 PA 9.34 8.01 9.74 5.82 4.33 5.73 4.20 1E−07 PA 5.925.67 5.88 6.90 2.33 2.35 2.33

[0161] TABLE 18 LPS Response as a Function of PA treatment LPS: S.I.10.00 5.00 1.00 0.50 0.10 0.05 0.01 1 PA 52.36 52.74 56.61 49.82 22.0018.14 8.47 0.1 PA 20.73 20.47 20.23 16.29 7.47 6.42 3.45 0.001 PA 13.8515.86 16.57 12.71 8.52 6.46 4.76 0.0001 PA 37.85 42.85 41.46 37.76 19.1015.78 6.41 1E−04 PA 23.10 37.92 32.05 27.29 13.38 20.52 7.49 1E−05 PA27.43 27.18 27.90 28.69 17.44 16.69 8.88 1E−06 PA 22.67 21.88 17.1417.60 12.40 9.27 6.30 1E−07 PA 14.49 16.56 17.95 18.56 10.99 7.20 3.40

[0162] Tables 15 to 18 show the effects of PA on the mitogen responsesof BXSB splenocytes. Although the kinetics are complex severalconclusions can be made. That PA (0.5 μG) elevates PHA and Con-Aresponses indicates normalization of T and B cell population dynamics.That PA also restores much of the amplitude of the mitogen responses andthe shape of the response curves indicates a partial or completerestoration of T cell and B cell response to mitogens.

[0163] In sum, the studies indicate that the aberrant BXSB spleniclymphocyte numbers as well as typical T and B mitogen responses can becorrected, at least in part, with PA treatment.

EXAMPLE 6

[0164] This example describes data indicating that PA reduces the amountof autoantibodies likely responsible for tissue destruction in BXSBanimals.

[0165] Humoral immunity is responsible for antibody production and issignificant as rheumatoid antibody are found in a significant percentageof RA patients. Abnormal antibodies have been found in the synovium ofsome patients. In the BXSB model, abnormal antibodies are produced.

[0166] Spleen cells obtained from control and PA treated BXSB animalswere studied in a plaque forming cell (PFC) assay. In this assay thetarget red blood cells are labeled with protein-A which will bind anysecreted immunoglobulin regardless of antigenic specificity thusproviding a broad view of humoral immunity. TABLE 19 BXSB Non−specificAntibody Producing Cells BXSB C57BL/6J Mean 60333 611.80 S.D. 33915658.67 N 45 75.00 SEM 5056 120.26

[0167] Table 19 shows the Ig-PFC responses of control BXSB splenocytes,which is 100× greater than control C57BL/6 splenocytes indicatingaberrant antibody production in the BXSB animals. TABLE 20 PA Treatmentand Ig-PFC Production in BXSB Animals BXSB RX 0.1 Control Rx 1 PA PA Rx0.01 PA Rx 0.001 PA Mean 60333 20741 35808 27909 30533 N 45 11 12 11 15S.E.M. 5056 6603 12010 2959 9581 P- 4.22E−05 0.04 5.16E−07 0.01 valuesRx Rx 0.0001 0.00001 Rx 0.000001 Rx 0.0000001 PA PA PA PA Mean 385012310 16278 11042 N 5 10 9 12 S.E.M. 696 2040 4068 1751 P- 7.33E−152.92E−12 3.11E−08 8.16E−13 values

[0168] Table 20 shows the dose related effect of PA on Ig-PFCproduction. All PA amounts significantly reduced the over-production ofIg-PFCs with 0.0001 μG PA showing the greatest reduction.

[0169] BXSB control splenocytes make and secrete antibodies at enormouslevels (61400±6435 PFC/1E06 ). This is approximately 100 times thevalues observed in C57BL/6J normal controls. PA treatment significantlyreduces auto-antibody levels in a dose and time dependent manner. TABLE21 PA and the Effect on Circulating Auto-antibodies in BXSB AnimalsControl Rx 0.01 PA  2 weeks 0.262 0.016 0.318 0.000  5 weeks 0.272 0.0290.360 −0.032 11 weeks 1.317 0.103 1.449 0.125 14 weeks 0.402 0.283 0.3570.206

[0170] Table 21 shows the development of auto-antibodies in BXSB animals(the higher numbers indicate greater amounts of circulatingautoantibodies). PA treatment reduces autoantibody levels at all timepoints measured. TABLE 22 The Effect of PA Concentration onAuto-antibody Production (ANA) Control Rx 1 PA Rx 0.1 PA Rx 0.01 PA Rx0.001 PA Mean 0.475 0.306 0.176 0.091 0.341 N 11 7 7 9 11 S.E.M 0.140.03 0.09 0.03 0.10 P-value 0.16 0.07 0.02 0.25 Rx Rx 0.0001 0.00001 RxRx 0.0000001 PA PA 0.000001 PA PA Mean 0.229 0.090 0.176 0.098 N 9 12 1215 SEM 0.04 0.06 0.03 0.03 P-value 0.02 0.02 0.08 0.02

[0171] Table 22 shows that PA-mediated reduction of autoantibodies isdose dependent. The greatest reduction occurs at 0.00001 μG.

[0172] In sum, PA regulates the number of non-specific antibodies,reducing the amount of damaging autoantibodies. That PA also reducesantibody producing cells in BXSB spleens correlates with this data.Thus, PA treatment restores, at least in part, humoral immunity.

EXAMPLE 7

[0173] This example describes data indicating that PA reducescytotoxicity response of BXSB mice, re-regulating this response to at ornear base-line levels.

[0174] The cellular component of the immune system is the primaryintegrator of function for the entire immune system, supplying theT-cells and B-cells in their various differentiated forms for bothrecognition and effector function. In the BXSB animal model there havebeen reports that the Cell Mediated Immune (CMI) system is intact.However, contrary to these reports the data described below indicatethat CMI is affected in the BXSB mouse.

[0175] BXSB mice, both control and treated with PA, were studied fortheir ability to recognize and lyse non-specific targets labeled withradio-labeled chromium. In brief, about 200 μl spleen cells (1×10⁷/ml)from control and treated BXSB mice were plated on a microtiter plate inRPMI medium and five two fold serial dilutions were made in RPMI media.P815 cells were radiolabeled with chromium (Cr⁵¹) and added to each wellof a 96 well plate, centrifuged for 12 minutes and then incubated for3-4 hours at 37° C. Cells were re-centrifuged and a 110 μl samplecounted for Cr⁵¹. A more detailed protocol is contained in CurrentProtocols in Immunology, 3.11, Assays for T cell Function. TABLE 23Cytotoxicity Response of BXSB and Normal Mice BXSB Rx 3 weeks BXSBControl C57BL/6J (acute) E/F % Specific % Specific % Specific RatioLysis S. D. Lysis S. D. Lysis S. D.  100:1 122.0 5.0 2.0 0.1 5.43 4.1  50:1 95.1 9.4 −1.2 0.5 4.94 4.4   25:1 93.8 10.3 −1.5 0.6 3.94 3.312.5:1 85.4 8.3 −1.3 0.5 3.04 3.1 6.25:1 50.5 35.1 −1.2 0.2 2.84 3.03.13:1 5.3 5.3 −2.5 0.3

[0176] Table 23 shows that normal mice (middle column) have a typicalbase line level of natural killer activity to the P-815 target labeledwith Cr⁵¹; the level is between 0 and 3% cytotoxicity at an effectortarget ratio of 100:1. The left column shows the cytotoxicity responseof BXSB mice to the same target; the level of cytotoxicity is extremelyhigh at over 50% at an effector/target ratio of 6:1. PA treatment ofBXSB over a 3 to 15 week period (right column) reduced cytotoxicity tobase line levels, i.e. 0-3% at E/T ratio of 100:1 without any regressionanalysis possible.

[0177] In sum, the studies indicate that PA treatment reduces theun-regulated BXSB cytotoxicity responses by a factor of 20, to at ornear control base-line levels. Thus, PA treatment restores, at least inpart, cellular immunity.

EXAMPLE 8

[0178] This example describes data indicating that PA regulatesexpression of CD markers reflecting the regulation of celldifferentiation, proliferation or apoptosis of cells of lymphoidlineage.

[0179] The expression of clustered determinants (CD markers) indicatethe differentiation status of lymphoid cells. PA specifically regulatesthese markers. This regulation has direct correlations with the datadescribed above. TABLE 24A CD Marker Profiles 69+4− 69+4+ 69−4+ 4+8−4+8+ 4−8+ 69+8− 69+8+ 69−8+ BL6 7.6+−1.4 7.6+−1.6 8.5+−2.9 7.4+−4.47.6+−1.7 18+−3.2 30+−2.3 5.0+−0.9 66+−2.7 control N 23 23 23 23 23 23 2323 23 BXSB 20+−4.1 13+−1.5 68+−5.1 4.2+−7.7 8.5+−1.3 49+−6.9 22+−5.77.8+−1.5 71+−6.9 2-10 P 0.009 0.017 0.006 0.002 0.34 0.0008 0.11 0.070.25 one-tail BXSB 44+−1.0 20+−1.3 36+−2.1 16+−1.3 14+−0.8 70+−1.123+−2.3 14+−0.7 64+−2.2 11-15 P 2.5E−12 4.8E−5 1.8E−9 1.7E−12 0.0023.3E−14 0.03 1.7E−6 0.3 one-tail

[0180] Table 24A shows data of a number of T-cell CD markers.Approximately 80% of normal splenocytes (BL6 control) are non-activated(CD69−) T-cells (CD69−, CD4+). This population decreases in both young(2-10 experimental weeks, and 10 to 18 weeks chronological age) and old(11-15 experimental weeks, and 19 to 23 weeks chronological age) BXSBanimals. There is also a kinetic effect: the 11-15 week BXSB animals aremuch more severely compromised in terms of their T-cell markers thanyounger BXSB animals. This correlates to decreased cellular function andincreased overall death rate. TABLE 24B CD Marker Profiles cont'd 19+45−19+45+ 19−45+ 80+25− 80+25+ 80−25+ C57BL/6J 3+−0.6 80+−2 18+−3 70+−313+−1 18+−1 BXSB 8+−4 60+−12 35+−11 65+−7 17+−2 18+−5  2-10 wks P N.S.N.S. N.S. N.S. N.S. N.S. [one-tail] BXSB 14+−7 64+−22 22+−15 61+−6 18+−221+−8 11-15 wks P N.S. N.S. N.S. N.S. N.S. N.S. [one-tail]

[0181] Table 24B shows data of a number of B-cell CD markers. The firstset of three markers are indicative of resting B-cells (i.e.non-activated) and the last three represent activated B cells. Thesedata show that the B-cell population is refractory to both the strainand the stage of the disease process in the BXSB mice. Thus, it appearsthat the primary cellular immune component of BXSB combinedimmunodeficiency disease process involves T-cells.

[0182] Graph 1 shows data from the effect of chronic treatment (from 3to 15 weeks) of BXSB mice with varying concentrations of PA (1E-03 to1E-07 μG of PA/injection). The first significant feature of this data isthat treatment of BXSB mice with PA regulates the population ofCD69−/CD4+ T-cells. The regulation is inter-related with the regulationof the other T-cell markers which is to be expected because one cellpopulation acts on others in the series (and other cell series) in bothfeedback and feed forward mechanisms. There is also a PA dose responseeffect on cell populations, consistent with the response described abovein the functional assays; there is a dose-time kinetic response as well.These data indicate that PA treatment does in fact regulate T celldifferentiation.

[0183] Graph 1 also shows data indicating that the E-03 dose of PArectifies the decrease in CD69-CD4+ observed in BXSB controls at earlytime points, although later losing this effect at longer time points ofchronic treatment. In contrast, the E-07 dose appears to be effective atboth short and long time points. These data are consistent with featuresof the Bio-Regulatory regimen: 1) The dose response curves are gaussian;and 2) the small amounts of PA that produce the effects suggest aprocess oriented target instead of a more traditional single effectortarget.

[0184] Table 25 shows the effect of chronic PA (1E-05 μG/dose)administered three times per week over a 6 and 9 week period comparedwith acute treatment at the same amount three times per week for asingle 3 week period followed by an additional 6 and 9 weeks withouttreatment. The acute treatment is effective in modulating the CD markerdisplay. Although complex the data demonstrate the inter-relationshipbetween the various markers; again the CD 8 series appear to be lestsensitive to PA than the CD4 or B-cell series. TABLE 25 Acute vs ChronicTreatment 0.0000 1 ug PA Percentage CD69+CD4− CD69+CD4+ CD4+CD69−CD4+CD8− CD4+CD8+ CD8+CD4− CD69+CD8− CD69+CD8+ CD8+CD69− BXSB Control19.73 12.45 67.50 41.85 8.48 49.24 21.53 7.80 70.67 6 wks acute 56.2522.42 21.34 17.64 7.68 73.59 24.91 5.56 69.54 9 wks Acute 63.37 18.8217.81 24.38 12.81 61.90 42.74 7.16 50.10 BL6 Control 7.58 7.63 84.6973.81 7.60 18.41 29.61 5.01 65.56 Rx Chronic 6 wks 10.90 83.69 5.39 4.6210.35 85.03 6.70 10.13 83.17 Rx Chronic 9 wks 46.20 23.09 30.45 16.6410.40 72.96 23.02 12.74 64.23

[0185] Table 26 shows a time profile of a single PA dose (1E-05 μG) onthe nine T-cell marker series used in these studies. Again, the doublepositive cells (destined for apoptosis) as well as the activation serieswith CD8 are relatively refractory to treatment. PA treatment doesmodulate the activation CD4 series and mature cytotoxic T-cells(CD8+CD4−). TABLE 26 Treatment with 0.00001 ug PA Percentage CD69+CD4−CD69+CD4+ CD4+CD69− CD4+CD8− CD4+CD8+ CD8+CD4− CD69+CD8− CD69+CD8+CD8+CD69−  3 wks 29.79 12.41 57.8 42.57 7.22 50.21 24.98 4.78 70.24  6wks 10.9 83.69 5.36 4.62 10.35 85.03 6.7 10.13 83.17  9 wks 46.2 23.0930.45 16.64 10.4 72.96 23.02 12.74 64.23 12 wks 55.87 19.68 24.27 12.0514.68 73.27 25.44 17.5 57.06 Untreated BL 6 7.58 7.63 84.68 73.8 7.618.41 29.61 5.01 65.55 Untreated BXSB 44.34 19.69 35.78 15.92 13.9170.16 22.54 13.69 63.76

[0186] In sum, the studies indicate that acute or chronic PA treatmentregulated T-cell CD markers in BXSB mice in a dose and kinetic dependentmanner, which correlate with the functional changes described abovewhich include partial restoration of normal mitogenic responses (Example5), reduction of autoantibody production (Example 6), and reduction ofcytotoxicity response (Example 7). Thus, PA regulates thedifferentiation sequence of cells of lymphoid lineage.

What is claimed is:
 1. A method for modulating an immune response in asubject, comprising administering to the subject a compositioncomprising an effective amount of a lymphocyte differentiation factorsufficient to modulate the immune response.
 2. The method of claim 2,wherein the lymphocyte differentiation factor comprises protein A (PA).3. A method for treating an immune dysfunction in a subject with or atrisk of an immune dysfunction, comprising administering to the subject acomposition comprising an effective amount of protein A (PA) sufficientto treat the immune dysfunction.
 4. The method of claim 3, wherein theimmune dysfunction comprises an autoimmune disorder.
 5. The method ofclaim 4, wherein the autoimmune disorder-comprises severe combinedimmunodeficiency (SCID), recombinase activating gene (RAG 1/2)deficiency, adenosine deaminase (ADA) deficiency, interleukin receptor γchain (γc) deficiency, Janus-associated kinase 3 (JAK3) deficiency,reticular dysgenesis, primary T cell immunodeficiency, DiGeorgesyndrome, Nude syndrome, T cell receptor deficiency, MHC class IIdeficiency, TAP-2 deficiency (MHC class I deficiency), ZAP70 tyrosinekinase deficiency, purine nucleotide phosphorylase (PNP) deficiency, anantibody deficiency, X-linked agammaglobulinemia (Bruton's tyrosinekinase deficiency), autosomal recessive agammaglobulinemia, Mu heavychain deficiency, surrogate light chain (γ5/14.1) deficiency, Hyper-IgMsyndrome, X-linked (CD40 ligand deficiency), Ig heavy chain genedeletion, IgA deficiency, deficiency of IgG subclasses with and withoutIgA deficiency, common variable immunodeficiency (CVID), antibodydeficiency with normal immunoglobulins, transient hypogammaglobulinemiaof infancy, interferon γ receptor (IFNGR1 or IFNGR2) deficiency,interleukin 12 deficiency, interleukin 12 receptor deficiency,immunodeficiency with thymoma, Wiskott-Aldrich syndrome (WAS proteindeficiency), ataxia telangiectasia (ATM deficiency), X-linkedlymphoproliferative syndrome (SH2D1A/SAP deficiency) and hyper IgEsyndrome.
 6. A method for reducing an inflammatory response in a subjectwith or at risk of an inflammatory response, comprising administering tothe subject a composition comprising an effective amount of protein A(PA) sufficient to reduce an inflammatory response.
 7. The method ofclaim 6, wherein the inflammatory response is chronic or acute.
 8. Themethod of claim 6, wherein the inflammatory response is at least in partmediated by an antibody.
 9. The method of claim 8, wherein the antibodycomprises one or more auto-antibodies.
 10. The method of claim 6,wherein the inflammatory response is at least in part mediated bycellular immunity.
 11. A method for treating inflammation in a subjectwith or at risk of inflammation comprising administering to the subjecta composition comprising an effective amount of protein A (PA)sufficient to reduce the inflammation.
 12. The method of claim 11,wherein the inflammation is chronic or acute.
 13. The method of claim11, wherein the inflammation is at least in part antibody or cellmediated.
 14. The method of claim 11, wherein the treatment results in areduction in severity of a symptom of inflammation.
 15. The method ofclaim 14, wherein the symptom comprises swelling, pain, headache, fever,nausea, skeletal joint stiffness, or tissue or cell damage.
 16. Themethod of claim 11, wherein the treatment results in inhibition ofantibody production or lymphoid cell proliferation.
 17. A method ofinhibiting tissue or cell damage in a subject caused by an inflammatoryresponse or inflammation, comprising administering to the subject acomposition comprising an effective amount of protein A (PA) sufficientto treat inhibiting tissue or cell damage caused by an inflammatoryresponse or inflammation.
 18. The method of claim 17, wherein the tissueor cell damage is caused by a chronic or acute inflammatory response orinflammation.
 19. The method of claim 17, wherein the inflammatoryresponse or inflammation is at least in part antibody or cell mediated.20. The method of claim 17, wherein the tissue or cell damage is presentin thymus, liver, kidney, spleen, skin, or a skeletal joint.
 21. Themethod of claim 20, wherein the skeletal joint comprises a knee, ankle,hip, shoulder, wrist, finger, toe, or elbow.
 22. The method of claim 17,wherein the treatment results in inhibiting or preventing further tissueor cell damage.
 23. A method of treating existing tissue or cell damagein a subject caused by an inflammatory response or inflammation,comprising administering to the subject a composition comprising aneffective amount of protein A (PA) sufficient to treat existing tissueor cell damage caused by an inflammatory response or inflammation. 24.The method of claim 23, wherein the existing tissue or cell damage iscaused by a chronic or acute inflammatory response or inflammation. 25.The method of claim 23, wherein the inflammatory response orinflammation is at least in part antibody or cell mediated.
 26. Themethod of claim 23, wherein the existing tissue or cell damage ispresent in thymus, liver, kidney, spleen, skin, or a skeletal joint. 27.The method of claim 26, wherein the skeletal joint comprises a knee,ankle, hip, shoulder, wrist, finger, toe, or elbow.
 28. The method ofclaim 23, wherein the treatment results in reversing tissue or celldamage.
 29. The method of claim 23, wherein the treatment results ininhibiting or preventing further tissue or cell damage.
 30. A method oftreating splenomegalia in a subject, comprising administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to treat splenomegalia.
 31. A method of inhibitingproliferation or survival of a splenocyte in a subject having or at riskof having undesirable splenocyte proliferation or survival, comprisingadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to inhibit proliferation or survivalof the splenocyte.
 32. A method of stimulating differentiation orapoptosis of a splenocyte in a subject having or at risk of havingundesirable splenocyte proliferation or apoptosis, comprisingadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to stimulate differentiation orapoptosis of the splenocyte.
 33. A method of reducing antibodyproduction by a splenocyte in a subject having or at risk of havingundesirable numbers of an antibody, comprising administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to reduce antibody production by a splenocyte.
 34. A methodof reducing numbers of an antibody producing splenocyte in a subjecthaving or at risk of having undesirable numbers of the splenocytes,comprising administering to the subject a composition comprising aneffective amount of protein A (PA) sufficient to reduce the antibodyproducing splenocyte.
 35. The method of any of claims 33 and 34, whereinthe antibody comprises one or more auto-antibodies.
 36. A method ofreducing natural killer (NK) cell cytotoxicity in a subject having or atrisk of having undesirable NK cell cytotoxicity, comprisingadministering to the subject a composition comprising an effectiveamount of protein A (PA) sufficient to reduce undesirable NK cellcytotoxicity.
 37. A method of inhibiting rejection of a transplantedcell, tissue or organ in a subject, comprising administering to thesubject a composition comprising an effective amount of protein A (PA)sufficient to inhibit rejection of a transplanted cell, tissue or organ.38. The method of claim 37, wherein PA is administered prior to,substantially contemporaneously with, or following transplanting thecell, tissue or organ.
 39. The method of claim 38, wherein thetransplanted cell, tissue or organ is an allograft or xenograft.
 40. Amethod of stimulating differentiation of lymphoid cells, comprisingcontacting one or more lymphoid cells in vitro, ex vivo or in vivo witha composition comprising an effective amount of protein A (PA)sufficient to stimulate differentiation of one or more lymphoid cells.41. The method of claim 40, wherein the lymphoid cell is a T or B cell.42. The method of any of claims 1, 3, 6, 11, 17, 23, 30-34, 36, 37 or40, wherein the effective amount is a dose of about 1 picogram to about1 microgram of PA.
 43. The method of any of claims 1, 3, 6, 11, 17, 23,30-34, 36, 37 or 40, wherein the effective amount is a dose of about 1picogram to about 1 microgram of PA.
 44. The method of any of claims 1,3, 6, 11, 17, 23, 30-34, 36, 37 or 40, wherein the effective amount is asingle dose of about 100 picograms to about 1000 picograms of PAadministered intermittently over about 1 to 15 weeks.
 45. The method ofany of claims 1, 3, 6, 11, 17, 23, 30-34, 36, 37 or 40, wherein theeffective amount is a single dose of about 1 picogram to about 1microgram administered of PA on alternating days over about 7 to 21days.
 46. A composition comprising a unit dosage form of PA sufficientto reduce an inflammatory response, inflammation in a subject.
 47. Thecomposition of claim 46, wherein the unit dosage form is from about0.5-5, 5-10, 10-20, 20-50, 50-100, 100-500 or 100-1000 picograms. 48.The composition of claim 47, further comprising a drug that reduces aninflammatory response or inflammation.
 49. A pharmaceutical compositioncomprising a unit dosage form of PA sufficient to reduce an inflammatoryresponse, inflammation in a subject.
 50. A kit comprising a unit dosageform of PA sufficient to reduce an inflammatory response, inflammationor tissue or cell damage caused by an inflammatory response orinflammation in a subject.
 51. A kit comprising a unit dosage form ofPA, and instructions for use.
 52. The kit of claim 50 or 51, furthercomprising a plurality of unit dosage forms of PA.
 53. The kit of claim50 or 51, further comprising a drug that reduces an inflammatoryresponse or inflammation.